Molecular Targets for Modulation of Dissociative and Associative States

ABSTRACT

Methods of modulating dissociative and associative states in a subject are provided. In particular, neuromodulation of rhythmic neural activity in the posteromedial cortex can be used to induce or inhibit dissociative states in a subject.

BACKGROUND OF THE INVENTION

Recent advances in large-scale high-speed recording and control of neuronal activity have enabled exploration of natural and causal neural circuit-dynamics spanning the mammalian brain. We considered that these technological advances constitute a newly-emerged opportunity to explore cellular implementation of altered behavioral states that may require a global perspective for understanding. Dissociation represents a paradigmatic example of such a state.

In dissociation (elicited by diverse causes including stress, epilepsy, dissociative drugs, or certain neuropsychiatric disorders), normal integration of cognitive processing is disrupted. A selective uncoupling can be observed, with affective/emotional responses dissociated from sensory percepts, and sense-of-self dissociated from body position/action. The underlying cellular and circuit mechanisms of this fascinating and debilitating state remain unknown, despite substantial basic and clinical significance.

SUMMARY OF THE INVENTION

Methods of modulating dissociative and associative states in a subject are provided. In particular, neuromodulation of rhythmic neural activity in the posteromedial cortex can be used to induce or inhibit dissociative states in a subject.

In one aspect, a method of inhibiting development of a dissociative state in a subject is provided, the method comprising inhibiting a retrosplenial cortex layer-5 neuron optogenetically, wherein rhythmic neural activity is inhibited in the posteromedial cortex of the subject.

In certain embodiments, inhibiting the retrosplenial cortex layer-5 neuron optogenetically comprises: introducing a recombinant polynucleotide encoding a light-responsive ion channel into the retrosplenial cortex layer-5 neuron, wherein the light-responsive ion channel is expressed in the retrosplenial cortex layer-5 neuron; and illuminating the light-responsive ion channel with light at a wavelength that activates the light-responsive ion channel, wherein conduction of ions by the light-responsive ion channel in response to absorption of light results in hyperpolarization and inhibition of the retrosplenial cortex layer-5 neuron.

In certain embodiments, the light-responsive ion channel is a light-responsive anion-conducting opsin or a light-responsive proton conductance regulator.

In certain embodiments, the light-responsive anion-conducting opsin conducts chloride ions (Cl⁻).

In certain embodiments, the anion-conducting opsin is an anion-conducting channelrhodopsin or halorhodopsin. Exemplary anion-conducting halorhodopsins include, without limitation, Natronomonas pharaonis halorhodopsin (NpHR) and variants thereof (e.g., enhanced NpHR (eNpHR), eNpHR2.0, eNpHR3.0). Exemplary anion-conducting channelrhodopsins include, without limitation, iC1C2, SwiChR, SwiChR++, and iC++.

In certain embodiments, the light-responsive proton conductance regulator is a bacteriorhodopsin or an archaerhodopsin. Exemplary light-responsive proton conductance regulators include, without limitation, Arch from Halorubrum sodomense, ArchT from Halorubrum sp., TP009 from Leptosphaeria maculans, and Mac from Leptosphaeria maculans.

In certain embodiments, the polynucleotide encoding the light-responsive ion channel is provided by a viral vector. In some embodiments, the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is stereotactically injected into the retrosplenial cortex. In some embodiments, the vector further comprises a neuron-specific promoter operably linked to the polynucleotide encoding the light-responsive ion channel. In some embodiments, expression of the light-responsive ion channel is inducible.

In certain embodiments, illuminating the light-responsive ion channel comprises delivering light from a light source to the light-responsive ion channel using a fiber-optic-based optical neural interface. In some embodiments, the light source is a solid-state diode laser.

In certain embodiments, the dissociative disorder is caused by a drug, a seizure, childhood psychological or physical trauma, abuse, sexual assault, or a dissociation-linked neuropsychiatric disorder such as, but not limited to, post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder.

In another aspect, a method of inducing dissociative symptoms in a subject is provided, the method comprising performing rhythmic optogenetic activation of a retrosplenial cortex layer-5 neuron, wherein rhythmic neural activity is induced in the posteromedial cortex of the subject.

In certain embodiments, performing rhythmic optogenetic activation of a retrosplenial cortex layer-5 neuron comprises: introducing a recombinant polynucleotide encoding a light-responsive ion channel into the retrosplenial cortex layer-5 neuron, wherein the light-responsive ion channel is expressed in the retrosplenial cortex layer-5 neuron; and rhythmically illuminating the light-responsive ion channel with light at a wavelength that activates the light-responsive ion channel, wherein conduction of ions by the light-responsive ion channel in response to absorption of light results in depolarization and activation of the retrosplenial cortex layer-5 neuron.

In certain embodiments, the light-responsive ion channel is a light-responsive cation-conducting opsin. In some embodiments, the light-responsive cation-conducting opsin conducts calcium cations (Ca²⁺). In some embodiments, the light-responsive cation-conducting opsin is a light-responsive cation-conducting channelrhodopsin such as, but not limited to, a Chlamydomonas reinhardtii channelrhodopsin or a Volvox carteri channelrhodopsin. Exemplary light-responsive cation-conducting channelrhodopsins include, without limitation, a Chlamydomonas reinhardtii channelrhodopsin-1 (ChR1), a Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2), a Volvox carterichannelrhodopsin-1 (VChR1), and a chimeric ChR1-VChR1 channelrhodopsin.

In certain embodiments, the polynucleotide encoding the light-responsive ion channel is provided by a viral vector such as, but not limited to, a lentiviral vector or an adeno-associated viral (AAV) vector. In some embodiments, the viral vector is stereotactically injected into the retrosplenial cortex. In some embodiments, the vector further comprises a neuron-specific promoter operably linked to the polynucleotide encoding the light-responsive ion channel. In some embodiments, the expression of the light-responsive ion channel is inducible.

In certain embodiments, rhythmically illuminating the light-responsive ion channel comprises delivering light from a light source to the light-responsive ion channel using a fiber-optic-based optical neural interface. In some embodiments, the light source is a solid-state diode laser. In some embodiments, rhythmically illuminating the light-responsive ion channel with light comprises repeatedly delivering light at the wavelength that activates the light-responsive ion channel with a frequency of about 2 Hz.

In certain embodiments, rhythmically illuminating the light-responsive ion channel with light comprises alternating between a stimulation interval and a non-stimulation interval, wherein the light at the wavelength that activates the light-responsive ion channel is delivered during the stimulation interval, and no light or light that is not at the wavelength that activates the light-responsive ion channel or that inhibits the light-responsive ion channel is delivered during the non-stimulation interval. In some embodiments, the stimulation interval is 100 milliseconds to 300 milliseconds, including any amount of time in between such as 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, or 300 milliseconds. In one embodiment, the stimulation interval is 250 milliseconds.

In certain embodiments, the subject has a disorder of association. Exemplary disorders of association include, without limitation, chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction, and dependency.

In another aspect, a method of inducing dissociative symptoms in a subject is provided, the method comprising: positioning an electrode to deliver electrical stimulation to a posteromedial cortex region of the brain of the subject; and applying electrical stimulation to the posteromedial cortex region via the electrode in a manner effective generate rhythmic neural activity in the posteromedial cortex of the subject.

In certain embodiments, the method comprises applying electrical stimulation to a right posteromedial cortex region or a left posteromedial cortex region.

In certain embodiments, the electrode is placed on a surface of the right posteromedial cortex region or the left posteromedial cortex region.

In certain embodiments, the electrode is placed within the right posteromedial cortex region or the left posteromedial cortex region.

In certain embodiments, the electrode is a non-brain penetrating surface electrode array or a brain-penetrating electrode array.

In certain embodiments, the electrical stimulation is delivered to a posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the subject.

In certain embodiments, the subject has a disorder of association.

In certain embodiments, electrical stimulation is applied with a current ranging from 2 mA to 10 mA, or any current in between such as 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, or 10 mA.

In certain embodiments, the electrical stimulation is applied for up to 2 seconds. In some embodiments, the electrical stimulation is applied for 1 second to 2 seconds, including any amount of time in between such as 1, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or 2 seconds.

In certain embodiments, the electrical stimulation is applied unilaterally or bilaterally.

In certain embodiments, the electrical stimulation is applied with a frequency of 50 Hz.

In certain embodiments, the method further comprises mapping the brain of the subject to identify an optimal location in the posteromedial cortex for positioning the electrode for delivery of the electrical stimulation to induce dissociative symptoms in the subject.

In another aspect, a method of modulating associative or dissociative symptoms in a subject is provided, the method comprising delivering electrical stimulation to a posteromedial cortex region of the brain of the subject that modulates rhythmic neural activity in the posteromedial cortex of the subject.

In certain embodiments, the electrical stimulation is delivered optogenetically, with an electrode, or by transcranial magnetic stimulation.

In certain embodiments, the electrical stimulation is delivered to a posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the subject.

In certain embodiments, the electrical stimulation is applied unilaterally or bilaterally.

In certain embodiments, the method further comprises mapping the brain of the subject to identify an optimal location in the posteromedial cortex for positioning the electrode for delivery of the electrical stimulation to modulate associative or dissociative symptoms in the subject.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1J. Multiregional widefield imaging of cortical activity reveals ketamine-induced retrosplenial rhythm. FIG. 1A) Widefield microscope schematic. FIG. 1B) Transparent-skull preparation (left); 488 nm-fluorescence image, Thy1-GCaMP6s animal (right); A=anterior, P=posterior. FIG. 1C) Neocortex atlas for alignment: Motor (MOT), Somatosensory (SS), Parietal (PP), Visual (VIS), Retrosplenial (RSP). FIG. 1D) Video sequence: 410 nm-corrected fluorescence across 1 second in Thy1-GCaMP6s mouse. RSP activation: 0.13 s and 0.8 s; deactivation 0.54 s. FIG. 1E) (top) Fluorescence traces from five regions before (blue) and 10 min after (red) 50 mg/kg ketamine injection. 10 s 410-nm corrected data. (bottom) Corresponding power spectral density (PSD) plots, minutes 10-11 post-injection (mean/s.e.m; n=5 mice). FIG. 1F) Frequency-content change after ketamine, n=5 mice. One-way ANOVA, repeated measures, F(4,16)=25.4, p<1e-4. Corrected two-sided paired t-tests (**p<0.01). Hedge's g effect-sizes (3.96,3.90,3.76,3.62). FIG. 1G) 1-3 Hz power in RSP across 55 min, with i.p. injection of 50 mg/kg ketamine 5 min into recording (widefield imaging of GCaMP6s). Mean for n=6 mice: thick trace. FIG. 1H) PCP, MK801, and memantine: mean 1-3 Hz power (n=5 mice per drug), corrected 2-sided paired t-tests (*p<0.05, **p<0.01, ns p>0.05), Hedge's g effect-sizes PCP(4.23,4.57,5.14,4.87), MK801(1.80,1.79,2.16,1.28,1.56), memantine(1.00,0.96,0.27,0.82). FIG. 1I) (left) Single-cell activity traces; two-photon microscopy from layer 2/3 or 5 RSP after ketamine injection. (right) Mean 1-3 Hz power before/after ketamine across cells; n=5 mice (two-sided paired t-test, p=0.74 layer 2/3, n=5 mice, Hedge's g effect-size=−0.46; p<0.001 layer 5, n=5 mice, Hedge's g effect-size=3.16). FIG. 1J) tdTomato fluorescence after recombination of TRAP2;Ai14 mice, after 50 mg/kg ketamine injections; dense labeling of layer 5 cells. Dark cells: tdTomato expressing. Scale:1 mm.

FIGS. 2A-2G. Retrosplenial in vivo electrophysiology. FIG. 2A) Spike-train rasters from all simultaneously-recorded single-units from deep RSP in ketamine-injected head-fixed mouse. Below, firing rate of units above (Hz, 10 ms bin). Red boxes: ON-states for bursts (Methods). FIG. 2B) Multi-Neuropixel recording session. Ketamine injected i.p. 20 min into recording via previously-implanted cannula. FIG. 2C) 3D-reconstruction: Neuropixels insertions (all 4 mice, 16 probe-insertions total); brain areas noted by changing colors along each probe. Four Neuropixels implanted/recorded simultaneously. FIG. 2D) Average firing-rate change per region between 8-12 min before and 8-12 min after ketamine injection. Cells included from all 4 sessions. #units for each region: x label. Mean/s.e.m (corrected two-sided t-test vs. 0). FIG. 2E) Average RSP correlation (mean regional activity) before/after ketamine injection. #units indicated for each region. Mean/s.e.m (corrected two-sided t-test vs. 0). FIG. 2F) Correlation-matrix between cells, grouped by region before/after ketamine injection; 4-Neuropixel recording in mouse. FIG. 2G) Z-scored mean-activity traces; three simultaneously-recorded thalamic nuclei and RSP (green). *p<0.05, **p<0.01, ***p<0.001

FIGS. 3A-3H. Ketamine- and optogenetically-elicited dissociation-like behavioral phenotype. FIG. 3A) Hot-plate stimulus-detection (reflexive paw-flick) vs. affective/emotional response (protective paw-licking). FIG. 3B) Rate of reflexive paw-flicks/licks in ketamine-injected mice. One-way ANOVA with repeated measures: flick, p=0.38; lick, p=5E-4. Corrected two-sided unpaired t-tests. n=5 mice/group. For each dose in order, Hedge's g effect-sizes for flick (0.16,0.26,0.77,0.77) and lick (−0.028,0.53,−1.43,−2.19). FIG. 3C) (Left) Fiber photometry traces from RSP: rhythm at 25 mg/kg but not 13 mg/kg. (Right) 10 min after injection, power at each dose across several mice (unpaired-t-test p=0.008, Glass's A=3.2). FIG. 3D) Widefield imaging dose-response: mean 1-3 Hz power for 13 and 25 mg/kg doses (n=4 mice, two-sided unpaired t-test, p<0.027). FIG. 3E) Hot-plate test: other drug classes. Each experimental group compared with saline (control) group via corrected Mann-Whitney U-test. For each drug in order, Hedge's g effect-sizes: flick (3.10,−2.97,−2.19,1.14,−2.16), Glass's A: lick (−15.2,−2.89,0.093,−0.40,−0.90). FIG. 3F) (Left) Open-field velocity (5 min). Ketamine, LSD and diazepam decreased velocity. Buprenorphine increased velocity. Each experimental group compared with saline (control) via corrected Mann-Whitney U-test. Hedge's g effect-sizes: (−6.14,1.02,4.98,0.20,−2.34,−4.77). (Right) Traces of body-position with saline or dissociative drugs. Gray line: full 5 min session, black line: 20 s tracking (beginning minute-2). FIG. 3G) Viral injections, illumination pattern, behavioral arms. AAV1-nEF-DIO-eNpHR3.0-p2a-ChR2-YFP (eNPAC) injected bilaterally into Rpb4-cre RSP. Illumination: 20 Hz-blue and constant-orange light, alternating every 250 ms. FIG. 3H) Hot-plate: Retrosplenial left to right, paw-flick: YFP/Rhythmic p=0.53, YFP/Rand p=0.48; Rhythmic/Random p=0.43; paw-lick: YFP/Rhythmic p=0.034, YFP/Random p=0.38; Rhythmic/Random p=0.20, corrected two-sided unpaired t-tests, n=8 YFP, 7 Rhythmic, 7 Random mice. Hedge's g effect-sizes for flick (−0.31,0.50,0.79), lick (−1.43,−0.45,0.80). Somatosensory paw-flicks (p=0.50), paw-licks (p=0.71); Hedge's g effect-size for flick:−0.35, lick:−0.19, Two-sided paired t-test, n=8 control, n=6 somatosensory eNPAC mice. *p<0.05; **p<0.01, ***p<0.001

FIGS. 4A-4F. RSP HCN1 channels required for ketamine induction of deep rhythm and dissociation-like behavior. FIG. 4A) Possible mechanisms underlying RSP rhythm. FIG. 4B) Confocal images: (left to right) RSP, cingulate-cortex, subiculum, and dorsal-thalamus cells, green=GFP, blue=DAPI. Scale 1 mm (right panel). FIG. 4C) (Left) AAVdj-CaMKIIa-eNpHR3.0-YFP injected bilaterally in dorsal-thalamus or subiculum; GCaMP6m expressed in RSP. (Right) 1-3 Hz relative power for pre-, during, and post-optogenetic-inhibition 2 min epochs beginning 10 min after ketamine injection. Normalized by PSD during the pre-epoch, and then linearly-corrected to account for decrease in oscillation-power across 6 min recording. Thalamus inhibition increased oscillation-power (two-sided paired t-test, YFP p=0.057 n=5 mice, subiculum p=0.91 n=5, thalamus p=0.003 n=7). Hedge's g effect-sizes YFP=0.54, subiculum=0.065, thalamus=2.90. FIG. 4D) Confocal images: immunohistochemistry with antibodies against (top) GRIN1 or (bottom) HCN1 in wild-type mice. HCN1 image reveals low HCN1+density in superficial RSP. FIG. 4E) (Left) Photometry in RSP. AAVs expressing Cre-recombinase and Cre-dependent GcaMP6 injected for local disruption of NMDA-receptor or HCN1-channel expression. (Middle) Photometry traces. (Right) Reduced ketamine-induced oscillation-power in GRIN1 and HCN1 knockout mice (corrected two-sided paired t-test for within-mouse comparison and corrected two-sided independent t-test for between genotype comparisons, n.s.: p>0.05, *p<0.05, **p<0.01, ***p<0.001, n=7 mice/group, Hedge's g effect-sizes (WT-pre/WT-post=3.22, GRIN1-pre/GRIN1-post=2.03, HCN1-pre/HCN1-post=1.02, WT-post/HCN1-post=−2.75, WT-post/GRIN1-post=−2.26, HCN1-post/GRIN1-post=1.24, WT-pre/HCN1-pre=0.318, WT-pre/GRIN1-pre=0.322, HCN1-pre/GRIN1-pre=0.09). FIG. 4F) Hot-plate test. Reflexive paw-flicks (corrected Mann-Whitney U-test p>0.1) and affective paw-licks (corrected Mann-Whitney U-test, wild-type-vs-GRIN1, p=0.56; wild-type-vs-HCN1, p<0.001). Hedge's g effect-sizes for flick (0.77,0.070) and lick (0.40,2.46).

FIGS. 5A-5F. Human posteromedial cortex rhythm and self-reported dissociation. FIG. 5A) Simultaneous sEEG and 3D-electrode locations in patient, 1 Os before seizure-onset. Contact number (e.g. RC3: R (right-hemisphere) C (electrode-letter) 3 (contact-number) and anatomical-region (e.g. posteromedial). Rhythm observed in PMC in both hemispheres across contacts (magenta) not other regions (black). Contacts on each electrode: dots. FIG. 5B) Comments of patient describing pre-seizure aura experience. Notably, symptoms of dissociation consistently described (APA 2013) can involve reproducible perceptions of depersonalization (feeling of being outside observer of one's body/thoughts) and derealization (feeling of being detached from surroundings). Supplementary note: interview transcript. FIG. 5C) Simultaneously-recorded sEEG traces during pre-seizure epoch; different seizure from (a). One contact per electrode shown in black. Deep PMC contact: magenta. Seizure onset determined by epileptologist: vertical line. FIG. 5D) sEEG traces from deep and superficial PMC during aura (red) or non-seizure (blue) epochs. Mean+/−s.e.m. in (top) right and (bottom) left regions across aura periods. FIG. 5E) 3-4 Hz power during aura (red) or non-seizure/aura period (blue). Mean across channels in each region; aura periods: black dot. FIG. 5F) (Left) Patient's electrode locations (colored dots). Electrodes in deep PMC accentuated with blue (right) and gray (left) indicators. Comments describing experience during electrical stimulation of deep PMC shown. (Right) fraction of time aura reported for each sham or electrical stimulation (6 mA).

DETAILED DESCRIPTION

Before the methods of modulating dissociative and associative states in a subject by neuromodulation of rhythmic neural activity in the posteromedial cortex are further described, it is to be understood that this invention is not limited to a particular method or composition described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

As used herein the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a neuron” includes a plurality of such neurons and reference to “an electrode” includes reference to one or more electrodes and equivalents thereof, known to those skilled in the art, and so forth.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

The term “about,” particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent.

The term “dissociative disorder” or “disorder of dissociation” as used herein refers to any condition or disease associated with producing a dissociative state in a subject. Dissociative disorders may have various underlying causes, including, but not limited to, a drug, a seizure (e.g., epileptic or nonepileptic), childhood psychological or physical trauma, abuse, a sexual assault, or a dissociation-linked neuropsychiatric disorder, such as, but not limited to, post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder.

The term “associative disorder” or “disorder of association” as used herein refers to any condition or disease associated with producing an undesirable state of association in a subject. Disorders of association include, without limitation, chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction/dependency, or any dysfunctional association with specific, recurrent, and/or negative thoughts causing morbidity or mortality, wherein disconnecting from the damaging thoughts is beneficial.

“Substantially purified” generally refers to isolation of a substance (compound, drug, inhibitor, agonist, antagonist, polynucleotide, protein, polypeptide, antibody) such that the substance comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises 50%, preferably 80%-85%, more preferably 90-95% of the sample. Techniques for purifying substances of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.

The terms “recipient”, “individual”, “subject”, “host”, and “patient”, are used interchangeably herein and refer to any mammalian subject for whom diagnosis, treatment, or therapy is desired, particularly humans. “Mammal” for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, sheep, goats, pigs, etc. Preferably, the mammal is human.

The term “user” as used herein refers to a person that interacts with a device and/system disclosed herein for performing one or more steps of the presently disclosed methods. The user may be the patient receiving treatment. The user may be a health care practitioner, such as, the patient's physician.

The terms “treatment”, “treating”, “treat” and the like are used herein to generally refer to obtaining a desired pharmacologic and/or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and/or may be therapeutic in terms of a partial or complete stabilization or cure for a disease and/or adverse effect attributable to the disease. The term “treatment” encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and/or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and/or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and/or symptom(s). Those in need of treatment include those already inflicted (e.g., those with a disorder of dissociation or association) as well as those in which prevention is desired (e.g., those with increased susceptibility to abnormal states of dissociation such as caused by epilepsy or a dissociation-linked neuropsychiatric disorder such as post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder; or those with increased susceptibility to abnormal states of association such as caused by chronic pain, depression, anxiety, or obsessive-compulsive disorder, etc.).

By “therapeutically effective dose or amount” of electrical stimulation that modulates rhythmic neural activity in the posteromedial cortex or posteromedial cortical activity (e.g., electrical stimulation delivered optogenetically or with an electrode) is intended an amount that, when the electrical stimulation is administered, as described herein, brings about a positive therapeutic response in the treatment of a disorder of dissociation or association. For example, a positive therapeutic response in the treatment of a disorder of dissociation may include a reduction in symptoms of dissociation, such as associated with a seizure or a dissociation-linked neuropsychiatric disorder such as post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder. A positive therapeutic response in the treatment of a disorder of association may include a reduction in symptoms of association, such as specific, recurrent, and/or negative thoughts that cause morbidity or mortality, associated with chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction/dependency, or other dysfunctional condition, wherein disconnecting from the damaging thoughts associated with the disorder is beneficial. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

“Neural activity” as used herein, may refer to electrical activity of a neuron (e.g., changes in membrane potential of the neuron), as well as indirect measures of the electrical activity of one or more neurons. Thus, neural activity may refer to changes in field potential, changes in intracellular ion concentration (e.g., intracellular calcium concentration), and changes in magnetic resonance induced by electrical activity of neurons, as measured by, e.g., blood oxygenation level dependent (BOLD) signals in functional magnetic resonance imaging.

Modulating Associative and Dissociative Symptoms

The present invention is based on the discovery of a novel therapeutic methodology for modulating associative or dissociative symptoms in a subject. The methods utilize delivery of electrical stimulation that modulates rhythmic neural activity in the posteromedial cortex of the subject. Without being bound by a particular theory, a posteromedial cortical rhythm is associated with states of dissociation. Therefore, direct or indirect neuromodulation of the posteromedial cortex may induce or suppress the rhythmic neural activity associated with dissociative symptoms.

In some embodiments, neuromodulation is used to suppress rhythmic neural activity in the posteromedial cortex for treating a dissociative disorder. The term “dissociative disorder” or “disorder of dissociation” as used herein refers to any condition or disease associated with producing a dissociative state in a subject. Dissociative disorders may have various underlying causes, including, but not limited to, drug use, seizures (e.g., epileptic or nonepileptic), childhood psychological or physical trauma, abuse, sexual assault, or a dissociation-linked neuropsychiatric disorder, such as, but not limited to, post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder.

In some embodiments, neuromodulation is used to induce rhythmic neural activity in the posteromedial cortex to produce a dissociative state in a subject who is suffering from painful or damaging symptoms of a disorder of association. For example, in subjects who have chronic pain, an overwhelming association with pain can persist even when the original source of the pain no longer exists (e.g., pain from a limb that has been amputated). In conditions such as depression, anxiety, obsessive-compulsive disorder, addiction and dependency, dysfunctional association with specific, recurrent, and/or negative thoughts can cause morbidity or mortality, wherein disconnecting from the damaging thoughts by generating a dissociative state in a subject is beneficial.

Methods of the present disclosure include neuromodulation of the posteromedial cortical regions (e.g., posterior cingulate, precuneus, and/or retrosplenial) and/or thalamus to treat disorders of dissociation or association. Neuromodulation can be achieved using electrical stimulation (e.g., from chronically implanted, clinically approved electrodes), transcranial magnetic stimulation, or focused ultrasound, among other techniques. For electrical stimulation, deep brain stimulators can be used. Cortical layers or cell-types may be targeted specifically with genetically encodable modulation techniques, such as optogenetics. Methods of neuromodulation are described in further detail below.

Optogenetics

Optogenetics is used to allow optical control of activation (i.e., depolarization) or inhibition (i.e., hyperpolarization) of neurons that have been genetically modified to express light-responsive ion channels. In some embodiments, the light-responsive ion channel is a naturally occurring or synthetic opsin that uses a retinal-based cofactor (e.g., all-trans retinal for the microbial opsins) to respond to light. For example, light-responsive cation-conducting opsins (e.g., channelrhodopsin that conducts Ca²⁺) can be used to activate or depolarize neurons. Light-responsive anion-conducting opsins (e.g., channelrhodopsin or halorhodopsin that conduct chloride ions) or light-responsive proton conductance regulators (e.g., bacteriorhodopsin or archaerhodopsin) can be used to inhibit or hyperpolarize neurons. The levels of retinoids present in a mammalian brain are usually sufficient for expressed opsins to function without supplementation of cofactors. For a description of optogenetics and its use in controlling neural activity, see, e.g., Aravanis et al. (2007) J Neural Eng 4:S143—S156, Arenkiel et al. (2007) Neuron 54: 205-218, Boyden et al. (2005) Nat Neurosci 8: 1263-1268, Chow et al. (2010) Nature 463: 98-10, Gradinaru et al. (2007) J Neurosci 27: 14231-14238, Gradinaru et al. (2008) Brain Cell Biol 36: 129-139, Gradinaru et al. (2010) Cell 141: 1-12, Li et al. (2005) Proc Natl Acad Sci 102: 17816-17821, Lin et al. 2009. Characterization of engineered channelrhodopsin variants with improved properties and kinetics. Biophys J 96: 1803-1814, Yizhar et al. (2011) Microbial opsins: A family of single-component tools for optical control of neural activity. Cold Spring Harbor Protoc, Zhang et al. (2007) Nat Methods 4: 139-141, Zhang et al. (2006) Nat Methods 3: 785-792, Zhang et al. (2007) Nature 446: 633-639, Zhang et al. (2008) Nat Neurosci 11: 631-633; and U.S. Pat. Nos. 10,914,803; 10,589,123; 10,583,309; 10,568,516; 10,568,307; 10,538,560; 10,478,499; 10,220,092; 10,196,431; 10,087,223; 10,052,383; 9,969,783; 9,878,176; 9,855,442; 9,757,587; 9,458,208; and 8,834,546; herein incorporated by reference in their entireties.

In some embodiments, a target neuron is genetically modified to express a light-responsive ion channel that, when stimulated by an appropriate light stimulus, hyperpolarizes or depolarizes the stimulated target neuron. The term “genetic modification” refers to a permanent or transient genetic change induced in a cell following introduction into the cell of a heterologous nucleic acid (i.e., nucleic acid exogenous to the cell). Genetic change (“modification”) can be accomplished by incorporation of the heterologous nucleic acid into the genome of the host cell, or by transient or stable maintenance of the heterologous nucleic acid as an extrachromosomal element. Where the cell is a eukaryotic cell, a permanent genetic change can be achieved by introduction of the nucleic acid into the genome of the cell. Suitable methods of genetic modification include the use of viral infection, transfection, conjugation, protoplast fusion, electroporation, particle gun technology, calcium phosphate precipitation, direct microinjection, and the like.

In some cases, a target cell that expresses a light-responsive polypeptide can be activated or inhibited upon exposure to light of varying wavelengths. In some cases, a target cell that expresses a light-responsive polypeptide is a neuronal cell that expresses a light-responsive polypeptide, and exposure to light of varying wavelengths results in depolarization or polarization of the neuron.

In some instances, the light-responsive polypeptide is a light-responsive ion channel polypeptide. The light-responsive ion channel polypeptides are adapted to allow one or more ions to pass through the plasma membrane of a target cell when the polypeptide is illuminated with light of an activating wavelength. Light-responsive proteins may be characterized as ion pump proteins, which facilitate the passage of a small number of ions through the plasma membrane per photon of light, or as ion channel proteins, which allow a stream of ions to freely flow through the plasma membrane when the channel is open. In some embodiments, the light-responsive polypeptide depolarizes the excitable cell when activated by light of an activating wavelength. In some embodiments, the light-responsive polypeptide hyperpolarizes the excitable cell when activated by light of an activating wavelength.

In some cases, a light-responsive polypeptide mediates a hyperpolarizing current in the target cell it is expressed in when the cell is illuminated with light. Non-limiting examples of light-responsive polypeptides capable of mediating a hyperpolarizing current can be found, e.g., in U.S. Pat. Nos. 9,359,449 and 9,175,095. Non-limiting examples of hyperpolarizing light-responsive polypeptides include NpHr, eNpHr2.0, eNpHr3.0, eNpHr3.1 or GtR3. In some cases, a light-responsive polypeptide mediates a depolarizing current in the target cell it is expressed in when the cell is illuminated with light. Non-limiting examples of depolarizing light-responsive polypeptides include “C1V1”, ChR1, VChR1, ChR2. Additional information regarding other light-responsive cation channels, anion pumps, and proton pumps can be found in U.S. Patent Application Publication No: 2009/0093403; and U.S. Pat. No. 9,359,449.

In some embodiments, the light-responsive polypeptide can be activated by blue light (e.g., in range of 490 nm-450 nm). In one embodiment, the light-responsive polypeptide can be activated by light having a wavelength of about 473 nm. In some embodiments, the light-responsive polypeptide can be activated by yellow light (e.g., in range of 590 nm-560 nm). In another embodiment, the light-responsive polypeptide can be activated by light having a wavelength of about 560 nm. In another embodiment, the light-responsive polypeptide can be activated by red light (e.g., in range of 700 nm-635 nm). In another embodiment, the light-responsive polypeptide can be activated by light having a wavelength of about 630 nm. In other embodiments, the light-responsive polypeptide can be activated by violet light (e.g., in range of 450 nm-400 nm). In one embodiment, light-responsive polypeptide can be activated by light having a wavelength of about 405 nm. In other embodiments, the light-responsive polypeptide can be activated by green light (e.g., in range of 560 nm-520 nm). In other embodiments, the light-responsive polypeptide can be activated by cyan light (e.g., in range of 520 nm-490 nm). In other embodiments, the light-responsive polypeptide can be activated by orange light (e.g., in range of 635 nm-590 nm). A person of skill in the art would recognize that each light-responsive polypeptide will have its own range of activating wavelengths.

In some cases, the regions of the brain with neurons containing a light-responsive polypeptide are illuminated using one or more optical fibers. The optical fiber may be configured in any suitable manner to direct a light emitted from a suitable source of light, e.g., a laser or light-emitting diode (LED) light source, to the region of the brain. The optical fiber may be any suitable optical fiber. In some cases, the optical fiber is a multimode optical fiber. The optical fiber may include a core defining a core diameter, where light from the light source passes through the core. The optical fiber may have any suitable core diameter. In some cases, the core diameter of the optical fiber is 10 mm or more, e.g., 20 mm or more, 30 mm or more, 40 mm or more, 50 mm or more, 60 mm or more, including 80 mm or more, and is 1,000 mm or less, e.g., 500 mm or less, 200 mm or less, 100 mm or less, including 70 mm or less. In some embodiments, the core diameter of the optical fiber is in the range of 10 to 1,000 mm, e.g., 20 to 500 mm, 30 to 200 mm, including 40 to 100 mm.

The optical fiber end that is implanted into the target region of the brain may have any suitable configuration suitable for illuminating a region of the brain with a light stimulus delivered through the optical fiber. In some cases, the optical fiber includes an attachment device at or near the distal end of the optical fiber, where the distal end of the optical fiber corresponds to the end inserted into the subject. In some cases, the attachment device is configured to connect to the optical fiber and facilitate attachment of the optical fiber to the subject, such as to the skull of the subject. Any suitable attachment device may be used. In some cases, the attachment device includes a ferrule, e.g., a metal, ceramic or plastic ferrule. The ferrule may have any suitable dimensions for holding and attaching the optical fiber.

In certain embodiments, methods of the present disclosure may be performed using any suitable electronic components to control and/or coordinate the various optical components used to illuminate the regions of the brain. The optical components (e.g., light source, optical fiber, lens, objective, mirror, and the like) may be controlled by a controller, e.g., to coordinate the light source illuminating the regions of the brain with light pulses. The controller may include a driver for the light source that controls one or more parameters associated with the light pulses, such as, but not limited to the frequency, pulse width, duty cycle, wavelength, intensity, etc. of the light pulses. The controllers may be in communication with components of the light source (e.g., collimators, shutters, filter wheels, moveable mirrors, lenses, etc.).

In some embodiments, the light-responsive polypeptides are activated by light pulses that can have a duration for any of about 1 millisecond (ms), about 2 ms, about 3, ms, about 4, ms, about 5 ms, about 6 ms, about 7 ms, about 8 ms, about 9 ms, about 10 ms, about 15 ms, about 20 ms, about 25 ms, about 30 ms, about 35 ms, about 40 ms, about 45 ms, about 50 ms, about 60 ms, about 70 ms, about 80 ms, about 90 ms, about 100 ms, about 200 ms, about 300 ms, about 400 ms, about 500 ms, about 600 ms, about 700 ms, about 800 ms, about 900 ms, about 1 sec, about 1.25 sec, about 1.5 sec, or about 2 sec, inclusive, including any times in between these numbers. In some embodiments, the light-responsive polypeptides are activated by light pulses that can have a light power density of any of about 0.05 mW/mm², about 0.1 mW/mm², about 0.25 mW/mm², about 0.5 mW/mm², about 0.75 mW/mm², about 1 mW/mm², about 2 mW/mm², about 3 mW/mm², about 4 mW/mm², about 5 mW/mm², about 6 mW/mm², about 7 mW/mm², about 8 mW/mm², about 9 mW/mm², about 10 mW/mm², about 20 mW/mm², about 50 mW/mm², about 100 mW/mm², about 250 mW/mm², about 500 mW/mm², about 750 mW/mm², about 1000 mW/mm², about 1100 mW/mm², about 1200 mW/mm², about 1300 mW/mm², about 1400 mW/mm², about 1500 mW/mm², about 1600 mW/mm², about 1700 mW/mm², about 1800 mW/mm², about 1900 mW/mm², about 2000 mW/mm², about 2100 mW/mm², about 2200 mW/mm², about 2300 mW/mm², about 2400 mW/mm², about 2500 mW/mm², about 2600 mW/mm², about 2700 mW/mm², about 2800 mW/mm², about 2900 mW/mm², about 3000 mW/mm², about 3100 mW/mm², about 3100 mW/mm², about 3300 mW/mm², about 3400 mW/mm², or about 3500 mW/mm², inclusive, including any values between these numbers.

The light stimulus used to activate the light-responsive polypeptide may include light pulses characterized by, e.g., frequency, pulse width, duty cycle, wavelength, intensity, etc. In some cases, the light stimulus includes two or more different sets of light pulses, where each set of light pulses is characterized by different temporal patterns of light pulses. The temporal pattern may be characterized by any suitable parameter, including, but not limited to, frequency, period (i.e., total duration of the light stimulus), pulse width, duty cycle, etc.

The light pulses may have any suitable frequency. In some cases, the set of light pulses contains a single pulse of light that is sustained throughout the duration of the light stimulus. In some cases, the light pulses of a set have a frequency of 0.1 Hz or more, e.g., 0.5 Hz or more, 1 Hz or more, 5 Hz or more, 10 Hz or more, 20 Hz or more, 30 Hz or more, 40 H or more, including 50 Hz or more, or 60 Hz or more, or 70 Hz or more, or 80 Hz or more, or 90 Hz or more, or 100 Hz or more, and have a frequency of 100,000 Hz or less, e.g., 10,000 Hz or less, 1,000 Hz or less, 500 Hz or less, 400 Hz or less, 300 Hz or less, 200 Hz or less, including 100 Hz or less. In some embodiments, the light pulses have a frequency in the range of 0.1 to 100,000 Hz, e.g., 1 to 10,000 Hz, 1 to 1,000 Hz, including 5 to 500 Hz, or 10 to 100 Hz.

In some cases, the two sets of light pulses are characterized by having different parameter values, such as different pulse widths, e.g. short or long. The light pulses may have any suitable pulse width. In some cases, the pulse width is 0.1 ms or longer, e.g., 0.5 ms or longer, 1 ms or longer, 3 ms or longer, 5 ms or longer, 7.5 ms or longer, 10 ms or longer, including 15 ms or longer, or 20 ms or longer, or 25 ms or longer, or 30 ms or longer, or 35 ms or longer, or 40 ms or longer, or 45 ms or longer, or 50 ms or longer, and is 500 ms or shorter, e.g., 100 ms or shorter, 90 ms or shorter, 80 ms or shorter, 70 ms or shorter, 60 ms or shorter, 50 ms or shorter, 45 ms or shorter, 40 ms or shorter, 35 ms or shorter, 30 ms or shorter, 25 ms or shorter, including 20 ms or shorter. In some embodiments, the pulse width is in the range of 0.1 to 500 ms, e.g., 0.5 to 100 ms, 1 to 80 ms, including 1 to 60 ms, or 1 to 50 ms, or 1 to 30 ms.

The average power of the light pulse, measured at the tip of an optical fiber delivering the light pulse to regions of the brain, may be any suitable power. In some cases, the power is 0.1 mW or more, e.g., 0.5 mW or more, 1 mW or more, 1.5 mW or more, including 2 mW or more, or 2.5 mW or more, or 3 mW or more, or 3.5 mW or more, or 4 mW or more, or 4.5 mW or more, or 5 mW or more, and may be 1,000 mW or less, e.g., 500 mW or less, 250 mW or less, 100 mW or less, 50 mW or less, 40 mW or less, 30 mW or less, 20 mW or less, 15 mW or less, including 10 mW or less, or 5 mW or less. In some embodiments, the power is in the range of 0.1 to 1,000 mW, e.g., 0.5 to 100 mW, 0.5 to 50 mW, 1 to 20 mW, including 1 to 10 mW, or 1 to 5 mW.

The wavelength and intensity of the light pulses may vary and may depend on the activation wavelength of the light-responsive polypeptide, optical transparency of the region of the brain, the desired volume of the brain to be illuminated, etc.

In some embodiments, rhythmic neural activity is induced in the posteromedial cortex of the subject by rhythmically illuminating a light-responsive polypeptide. For example, light delivery may alternate between a stimulation interval and a non-stimulation interval, wherein the light at the wavelength that activates the light-responsive polypeptide (e.g., light-responsive ion channel) is delivered during the stimulation interval, and no light or light that is not at the wavelength that activates the light-responsive polypeptide (e.g., light-responsive ion channel) or that inhibits the light-responsive polypeptide (e.g., light-responsive ion channel) is delivered during the non-stimulation interval.

The volume of a brain region illuminated by the light pulses may be any suitable volume. In some cases, the illuminated volume is 0.001 mm³ or more, e.g., 0.005 mm³ or more, 0.001 mm³ or more, 0.005 mm³ or more, 0.01 mm³ or more, 0.05 mm³ or more, including 0.1 mm³ or more, and is 100 mm³ or less, e.g., 50 mm³ or less, 20 mm³ or less, 10 mm³ or less, 5 mm³ or less, 1 mm³ or less, including 0.1 mm³ or less. In certain cases, the illuminated volume is in the range of 0.001 to 100 mm³, e.g., 0.005 to 20 mm³, 0.01 to 10 mm³, 0.01 to 5 mm³, including 0.05 to 1 mm³.

In some embodiments, the light-responsive polypeptide expressed in a cell can be fused to one or more amino acid sequence motifs selected from the group consisting of a signal peptide, an endoplasmic reticulum (ER) export signal, a membrane trafficking signal, and/or an N-terminal golgi export signal. The one or more amino acid sequence motifs which enhance light-responsive protein transport to the plasma membranes of mammalian cells can be fused to the N-terminus, the C-terminus, or to both the N- and C-terminal ends of the light-responsive polypeptide. In some cases, the one or more amino acid sequence motifs which enhance light-responsive polypeptide transport to the plasma membranes of mammalian cells is fused internally within a light-responsive polypeptide. Optionally, the light-responsive polypeptide and the one or more amino acid sequence motifs may be separated by a linker. In some embodiments, the light-responsive polypeptide can be modified by the addition of a trafficking signal (ts) which enhances transport of the protein to the cell plasma membrane. In some embodiments, the trafficking signal can be derived from the amino acid sequence of the human inward rectifier potassium channel Kir2.1. In some embodiments, the signal peptide sequence in the protein can be deleted or substituted with a signal peptide sequence from a different protein.

Exemplary light-responsive polypeptides and amino acid sequence motifs that find use in the present system and method are disclosed in, e.g., U.S. Pat. Nos. 10,538,560; 10,568,307; 9,284,353; 9,359,449; and 9,365,628; herein incorporated by reference.

Light-responsive polypeptides of interest include, for example, a step function opsin (SFO)6 protein or a stabilized step function opsin (SSFO) protein that can have specific amino acid substitutions at key positions in the retinal binding pocket of the protein. See, for example, WO 2010/056970, the disclosure of which is hereby incorporated by reference in its entirety. The polypeptide may be a cation channel derived from Volvox carteri (VChR1), optionally comprising one or more amino acid substitutions, e.g., C123A; C123S; D151A, etc. A light-responsive cation channel protein can be a C1V1 chimeric protein derived from the VChR1 protein of Volvox carteri and the ChR1 protein from Chlamydomonas reinhardti, wherein the protein comprises the amino acid sequence of VChR1 having at least the first and second transmembrane helices replaced by the first and second transmembrane helices of ChR1, optionally having an amino acid substitution at amino acid residue E122 or E162. In other embodiments, the light-responsive cation channel protein is a C1C2 chimeric protein derived from the ChR1 and the ChR2 proteins from Chlamydomonas reinhardti, wherein the protein is responsive to light and is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments, a depolarizing light-responsive polypeptide is a red shifted variant of a depolarizing light-responsive polypeptide derived from Chlamydomonas reinhardtii; referred to as a “ReaChR polypeptide” or “ReaChR protein” or “ReaChR.” In some embodiments, a depolarizing light-responsive polypeptide is a SdChR polypeptide derived from Scherffelia dubia, wherein the SdChR polypeptide is capable of transporting cations across a cell membrane when the cell is illuminated with light. In some embodiments, a depolarizing light-responsive polypeptide is CnChR1, derived from Chlamydomonas noctigama, wherein the CnChR1 polypeptide is capable of transporting cations across a cell membrane when the cell is illuminated with light. In some embodiments, the light-responsive cation channel protein is a CsChrimson chimeric protein derived from a CsChR protein of Chloromonas subdivisa and CnChR1 protein from Chlamydomonas noctigama, wherein the N-terminus of the protein comprises the amino acid sequence of residues 1-73 of CsChR followed by residues 79-350 of the amino acid sequence of CnChR1; is responsive to light; and is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments, a depolarizing light-responsive polypeptide can be, e.g., ShChR1, derived from Stigeoclonium helveticum, wherein the ShChR1 polypeptide is capable of transporting cations across a cell membrane when the cell is illuminated with light.

In some embodiments, a depolarizing light-responsive polypeptide is derived from Chlamydomonas reinhardtii (CHR1, and particularly CHR2) wherein the polypeptide is capable of transporting cations across a cell membrane when the cell is illuminated with light; and is capable of mediating a depolarizing current in the cell when the cell is illuminated with light. In some embodiments CaMKIIa-driven, humanized channelrhodopsin CHR2 H134R mutant fused to EYFP is used for optogenetic activation. The light used to activate the light-responsive cation channel protein derived from Chlamydomonas reinhardtii can have a wavelength between about 460 and about 495 nm or can have a wavelength of about 480 nm. The light-responsive cation channel protein can additionally comprise substitutions, deletions, and/or insertions introduced into a native amino acid sequence to increase or decrease sensitivity to light, increase or decrease sensitivity to particular wavelengths of light, and/or increase or decrease the ability of the light-responsive cation channel protein to regulate the polarization state of the plasma membrane of the cell. Additionally, the light-responsive cation channel protein can comprise one or more conservative amino acid substitutions and/or one or more non-conservative amino acid substitutions. The light-responsive proton pump protein containing substitutions, deletions, and/or insertions introduced into the native amino acid sequence suitably retains the ability to transport cations across a cell membrane. The protein may comprise various amino acid substitutions, e.g., one or more of H134R; T159C; L132C; E123A; etc. The protein may further comprise a fluorescent protein, for example, but not limited to, a yellow fluorescent protein, a red fluorescent protein, a green fluorescent protein, or a cyan fluorescent protein.

Neurons can be selectively activated or inhibited optogenetically by engineering neurons to express one or more light-responsive polypeptides configured to hyperpolarize or depolarize the neurons. Suitable light-responsive polypeptides and methods used thereof are described further below.

A light-responsive polypeptide for use in the present disclosure may be any suitable light-responsive polypeptide for selectively activating neurons of a subtype by illuminating the neurons with an activating light stimulus. In some instances, the light-responsive polypeptide is a light-responsive ion channel polypeptide. The light-responsive ion channel polypeptides are adapted to allow one or more ions to pass through the plasma membrane of a target cell when the polypeptide is illuminated with light of an activating wavelength. Light-responsive proteins may be characterized as ion pump proteins, which facilitate the passage of a small number of ions through the plasma membrane per photon of light, or as ion channel proteins, which allow a stream of ions to freely flow through the plasma membrane when the channel is open. In some embodiments, the light-responsive polypeptide depolarizes the cell when activated by light of an activating wavelength. In some embodiments, the light-responsive polypeptide hyperpolarizes the cell when activated by light of an activating wavelength. Suitable hyperpolarizing and depolarizing polypeptides are known in the art and include, e.g., a channelrhodopsin (e.g., ChR2), variants of ChR2 (e.g., C128S, D156A, C128S+D156A, E123A, E123T), iC1C2, C1C2, GtACR2, NpHR, eNpHR3.0, C1V1, VChR1, VChR2, SwiChR, Arch, ArchT, KR2, ReaChR, ChiEF, Chronos, ChRGR, CsChrimson, and the like. In some cases, the light-responsive polypeptide includes bReaCh-ES, as described in, e.g., Rajasethupathy et al., Nature. 2015 Oct. 29; 526(7575):653, which is incorporated by reference. Hyperpolarizing and depolarizing opsins have been described in various publications; see, e.g., Berndt and Deisseroth (2015) Science 349:590; Berndt et al. (2014) Science 344:420; and Guru et al. (Jul. 25, 2015) Intl. J. Neuropsychopharmacol. pp. 1-8 (PMID 26209858).

The light-responsive polypeptide may be introduced into the neurons using any suitable method. In some cases, the neurons of a subtype of interest are genetically modified to express a light-responsive polypeptide. In some cases, the neurons may be genetically modified using a viral vector, e.g., an adeno-associated viral vector, containing a nucleic acid having a nucleotide sequence that encodes the light-responsive polypeptide. The viral vector may include any suitable control elements (e.g., promoters, enhancers, recombination sites, etc.) to control expression of the light-responsive polypeptide according to neuronal subtype, timing, presence of an inducer, etc.

“Operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a nucleotide sequence (e.g., a protein coding sequence, e.g., a sequence encoding an mRNA; a non-protein coding sequence, e.g., a sequence encoding a light-reactive protein; and the like) if the promoter affects its transcription and/or expression.

Neuron-specific promoters and other control elements (e.g., enhancers) are known in the art. Suitable neuron-specific control sequences include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956; see also, e.g., U.S. Pat. Nos. 6,649,811, 5,387,742); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51:7-19; and Llewellyn et al. (2010) Nat. Med. 16:1161); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Nucl. Acids. Res. 15:2363-2384 (1987) and Neuron 6:583-594 (1991)); a GnRH promoter (see, e.g., Radovick et al., Proc. Natl. Acad. Sci. USA 88:3402-3406 (1991)); an L7 promoter (see, e.g., Oberdick et al., Science 248:223-226 (1990)); a DNMT promoter (see, e.g., Bartge et al., Proc. Natl. Acad. Sci. USA 85:3648-3652 (1988)); an enkephalin promoter (see, e.g., Comb et al., EMBO J. 17:3793-3805 (1988)); a myelin basic protein (MBP) promoter; a CMV enhancer/platelet-derived growth factor-.beta. promoter (see, e.g., Liu et al. (2620) Gene Therapy 11:52-60); a motor neuron-specific gene Hb9 promoter (see, e.g., U.S. Pat. No. 7,632,679; and Lee et al. (2620) Development 131:3295-3306); and an alpha subunit of Ca²⁺-calmodulin-dependent protein kinase II (CaMKII) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93:13250). Other suitable promoters include elongation factor (EF) 1 and dopamine transporter (DAT) promoters.

In some cases, neuronal subtype-specific expression of the light-responsive polypeptide may be achieved by using recombination systems, e.g., Cre-Lox recombination, Flp-FRT recombination, etc. Cell type-specific expression of genes using recombination has been described in, e.g., Fenno et al., Nat Methods, 2014 July; 11(7):763; and Gompf et al., Front Behav Neurosci. 2015 Jul. 2; 9:152, which are incorporated by reference herein.

In some embodiments, the vector is a recombinant adeno-associated virus (AAV) vector. AAV vectors are DNA viruses of relatively small size that can integrate, in a stable and site-specific manner, into the genome of the cells that they infect. They are able to infect a wide spectrum of cells without inducing any effects on cellular growth, morphology or differentiation, and they do not appear to be involved in human pathologies. The AAV genome has been cloned, sequenced and characterized. It encompasses approximately 4700 bases and contains an inverted terminal repeat (ITR) region of approximately 145 bases at each end, which serves as an origin of replication for the virus. The remainder of the genome is divided into two essential regions that carry the encapsidation functions: the left-hand part of the genome, that contains the rep gene involved in viral replication and expression of the viral genes; and the right-hand part of the genome, that contains the cap gene encoding the capsid proteins of the virus.

The application of AAV as a vector for gene therapy has been rapidly developed in recent years. Wild-type AAV could infect, with a comparatively high titer, dividing or non-dividing cells, or tissues of mammal, including human, and also can integrate into in human cells at specific site (on the long arm of chromosome 19) (Kotin et al, Proc. Natl. Acad. Sci. U.S.A., 1990. 87: 2211-2215; Samulski et al, EMBO J., 1991. 10: 3941-3950 the disclosures of which are hereby incorporated by reference herein in their entireties). AAV vector without the rep and cap genes loses specificity of site-specific integration, but may still mediate long-term stable expression of exogenous genes. AAV vector exists in cells in two forms, wherein one is episomic outside of the chromosome; another is integrated into the chromosome, with the former as the major form. Moreover, AAV has not hitherto been found to be associated with any human disease, nor any change of biological characteristics arising from the integration has been observed. There are sixteen serotypes of AAV reported in literature, respectively named AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16, wherein AAV5 is originally isolated from humans (Bantel-Schaal, and H. zur Hausen. Virology, 1984. 134: 52-63), while AAV1-4 and AAV6 are all found in the study of adenovirus (Ursula Bantel-Schaal, Hajo Delius and Harald zur Hausen. J. Virol., 1999. 73: 939-947).

AAV vectors may be prepared using any convenient methods. Adeno-associated viruses of any serotype are suitable (See, e.g., Blacklow, pp. 165-174 of “Parvoviruses and Human Disease” J. R. Pattison, ed. (1988); Rose, Comprehensive Virology 3:1, 1974; P. Tattersall “The Evolution of Parvovirus Taxonomy” In Parvoviruses (J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 5-14, Hudder Arnold, London, U K (2006); and D E Bowles, J E Rabinowitz, R J Samulski “The Genus Dependovirus” (J R Kerr, S F Cotmore. M E Bloom, R M Linden, C R Parrish, Eds.) p 15-23, Hudder Arnold, London, UK (2006), the disclosures of which are hereby incorporated by reference herein in their entireties). Methods for purifying for vectors may be found in, for example, U.S. Pat. Nos. 6,566,118, 6,989,264, and 6,995,006 and WO/1999/011764 titled “Methods for Generating High Titer Helper-free Preparation of Recombinant AAV Vectors”, the disclosures of which are herein incorporated by reference in their entirety. Preparation of hybrid vectors is described in, for example, PCT Application No. PCT/US2005/027091, the disclosure of which is herein incorporated by reference in its entirety. The use of vectors derived from the AAVs for transferring genes in vitro and in vivo has been described (See e.g., International Patent Application Publication Nos: 91/18088 and WO 93/09239; U.S. Pat. Nos. 4,797,368, 6,596,535, and 5,139,941; and European Patent No: 0488528, all of which are herein incorporated by reference in their entirety). These publications describe various AAV-derived constructs in which the rep and/or cap genes are deleted and replaced by a gene of interest, and the use of these constructs for transferring the gene of interest in vitro (into cultured cells) or in vivo (directly into an organism). The replication defective recombinant AAVs according to the invention can be prepared by co-transfecting a plasmid containing the nucleic acid sequence of interest flanked by two AAV inverted terminal repeat (ITR) regions, and a plasmid carrying the AAV encapsidation genes (rep and cap genes), into a cell line that is infected with a human helper virus (for example an adenovirus). The AAV recombinants that are produced are then purified by standard techniques.

In some embodiments, the vector(s) for use in the methods of the invention are encapsidated into a virus particle (e.g., AAV virus particle including, but not limited to, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, and AAV16). Accordingly, the invention includes a recombinant virus particle (recombinant because it contains a recombinant polynucleotide) comprising any of the vectors described herein. Methods of producing such particles are known in the art and are described in U.S. Pat. No. 6,596,535.

It is understood that one or more vectors may be administered to neural cells. If more than one vector is used, it is understood that they may be administered at the same or at different times.

Delivery of Electrical Stimulation with an Electrode

In some embodiments, electrical stimulation is applied to the posteromedial cortex region using an electrode in a manner effective to modulate rhythmic neural activity in the posteromedial cortex of a subject. The subject methods include applying electrical stimulation to the right posteromedial cortex region and/or left posteromedial cortex region of brain of the subject. The method may include a step of positioning an electrode at a posteromedial cortex region of the brain such as within the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. One or more electrodes may be positioned at the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. The electrode may be non-brain penetrating surface electrode(s) or brain-penetrating depth electrode(s). The electrical stimulation may be applied in a manner effective for modulating rhythmic neural activity in the posteromedial cortex of the subject. In some embodiments, the electrical stimulation is applied in a manner effective to generate rhythmic neural activity in the posteromedial cortex of the subject. In other embodiments, the electrical stimulation is applied in a manner effective to suppress rhythmic neural activity in the posteromedial cortex of the subject.

The electrical stimulation may be applied using a single electrode, electrode pairs, or an electrode array. Electrical stimulation may be applied unilaterally to the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the brain. In certain embodiments, electrical stimulation may be applied bilaterally. Bilateral electrical stimulation may be simultaneous or sequential. In embodiments where the electrical stimulation is applied at least two times, the site to which the electrical stimulation is applied may be alternated or otherwise spatially or temporally patterned.

Positioning an electrode for applying electrical stimulation at the specified region(s) of the brain may be carried out using standard surgical procedures for placement of intra-cranial electrodes. As used herein, the phrases “an electrode” or “the electrode” refer to a single electrode or multiple electrodes such as an electrode array. As used herein, the term “contact” as used in the context of an electrode in contact with a region of the brain refers to a physical association between the electrode and the region. In other words, an electrode that is in contact with a region of the brain is physically touching the region of the brain. An electrode in contact with a region of the brain can conduct electricity into the brain. Electrodes used in the methods disclosed herein may be monopolar (cathode or anode) or bipolar (e.g., having an anode and a cathode). The electrode(s) used for applying electric stimulation to the specified region(s) of the brain are also referred to as stimulation electrode(s).

In certain cases, placing the electrode at the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex may involve positioning the electrode on the surface of the specified region(s) of the brain. The electrode may be placed on surface of brain at the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. The electrode may contact at least a portion of the surface of the brain at the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. In some embodiments, the electrode may contact substantially the entire surface area at the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. In some embodiments, the electrode may additionally contact area(s) adjacent to the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. In some embodiments, an electrode array arranged on a planar support substrate may be used for electrically stimulating one or more regions of brains as specified herein. The surface area of the electrode array may be determined by the desired area of contact between the electrode array and the brain. An electrode for implanting on a brain surface, such as, a surface electrode or a surface electrode array may be obtained from a commercial supplier. A commercially obtained electrode/electrode array may be modified to achieve a desired contact area. In some cases, the non-brain penetrating electrode (also referred to as surface electrode) that may be used in the methods disclosed herein may be an electrocorticography (ECoG) electrode or an electroencephalography (EEG) electrode.

In certain cases, placing the electrode at a target area or site (i.e., the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex) may involve positioning a brain penetrating electrode (also referred to as depth electrode) in the specified region(s) of the brain. The electrode may be placed in the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. In some embodiments, the electrode may additionally contact area(s) adjacent to the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. In some embodiments, an electrode array may be used for electrically stimulating one or both regions of brains as specified herein. The depth to which the electrode is inserted into the brain may be determined by the desired level of contact between the electrode array and the brain. A brain-penetrating electrode array may be obtained from a commercial supplier. A commercially obtained electrode array may be modified to achieve a desired depth of insertion into the brain tissue.

In certain embodiments of the present methods, electrical stimulation is applied only to the posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex. In other embodiments, electrical stimulation is applied to one or more regions in the posterior cingulate, precuneus, and/or retrosplenial region of the posteromedial cortex and/or one or more additional regions in the brain such as the thalamus. In some embodiments, electrical stimulation is applied to the anterior thalamus. In some embodiments, electrical stimulation is applied to one or more thalamic nuclei (e.g., lateral dorsal, anteroventral).

The precise number of electrodes contained in an electrode array may vary. In certain aspects, an electrode array may include two or more electrodes, such as 3 or more, including 4 or more, e.g., about 3 to 6 electrodes, about 6 to 12 electrodes, about 12 to 18 electrodes, about 18 to 24 electrodes, about 24 to 30 electrodes, about 30 to 48 electrodes, about 48 to 72 electrodes, about 72 to 96 electrodes, or about 96 or more electrodes. The electrodes may be arranged into a regular repeating pattern (e.g., a grid, such as a grid with about 1 cm spacing between electrodes), or no pattern. An electrode that conforms to the target site for optimal delivery of electrical stimulation may be used. One such example, is a single multi contact electrode with eight contacts separated by 2½ mm. Each contract would have a span of approximately 2 mm. Another example is an electrode with two 1 cm contacts with a 2 mm intervening gap. Yet further, another example of an electrode that can be used in the present methods is a 2 or 3 branched electrode to cover the target site. Each one of these three pronged electrodes has four 1-2 mm contacts with a center to center separation of 2 of 2.5 mm and a span of 1.5 mm.

The size of each electrode may also vary depending upon such factors as the number of electrodes in the array, the location of the electrodes, the material, the age of the patient, and other factors. In certain aspects, an electrode array has a size (e.g., a diameter) of about 5 mm or less, such as about 4 mm or less, including 4 mm-0.25 mm, 3 mm-0.25 mm, 2 mm-0.25 mm, 1 mm-0.25 mm, or about 3 mm, about 2 mm, about 1 mm, about 0.5 mm, or about 0.25 mm.

In certain embodiments, the method further comprises mapping the brain of the subject to optimize positioning of an electrode for applying electrical stimulation. Positioning of a stimulation electrode is optimized to maximize clinical responses to electrical stimulation, to modulate rhythmic neural activity in the posteromedial cortex of a subject, and to relieve symptoms of a disorder of association or dissociation. In some embodiments, one or more regions of the posteromedial cortex are mapped to determine optimal positioning of stimulation electrodes. For example, the posterior cingulate, precuneus, and/or retrosplenial region of the posteromedial cortex may be mapped to determine optimal positioning of stimulation electrodes. In some embodiments, a subject's brain activity (e.g., at one or more regions in the posteromedial cortex) may be measured prior to the application of electrical stimulation to the posteromedial cortex and assessing may include comparing the rhythmic neural activity in the posteromedial cortex after applying electrical stimulation to that before applying electrical stimulation and a change in the post-treatment brain activity (e.g., inhibiting or inducing rhythmic neural activity in the posteromedial cortex) may indicate successful treatment. The effectiveness of electrical stimulation at a particular site may be further assessed by neurological examinations to determine whether dissociation is induced or suppressed by applying the electrical stimulation. A subject may be evaluated to determine whether the electrical stimulation ameliorates symptoms of a disorder of association or dissociation. In certain cases, an interviewer-administered assessment may be used alone or in conjunction with a self-reporting tool to determine if electrical stimulation relieves symptoms of a disorder of association or dissociation.

As set forth herein, the method involves applying electrical stimulation to the posteromedial cortex in a manner effective to inhibit or generate rhythmic neural activity. The parameters for applying the electrical stimulation may be determined empirically during treatment or may be pre-defined, such as, from a clinical study. The parameters of the electrical stimulation may include one or more of frequency, pulse width/duration, duty cycle, intensity/amplitude, pulse pattern, program duration, program frequency, and the like.

Frequency refers to the pulses produced per second during stimulation and is stated in units of Hertz (Hz, e.g., 50 Hz=50 pulses per second). The frequencies of electrical stimulation used in the present methods may vary widely depending on the numerous factors and may be determined empirically during treatment of the subject or may be pre-defined. In certain embodiments, the method may involve applying an electrical stimulation at a frequency of 10 Hz-500 Hz, such as, 10 Hz-300 Hz, 10 Hz-200 Hz, 10 Hz-150 Hz, 10 Hz-125 Hz, 10 Hz-100 Hz, 15 Hz-200 Hz, 15 Hz-300 Hz, 20 Hz-200 Hz, 25 Hz-400 Hz, 25 Hz-300 Hz, 25 Hz-200 Hz, 25 Hz-150 Hz, 25 Hz-100 Hz, 50 Hz-500 Hz, 50 Hz-400 Hz, 50 Hz-300 Hz, 50 Hz-200 Hz, 50 Hz-150 Hz, 50 Hz-100 Hz, 50 Hz-75 Hz, 75 Hz-300 Hz, 75 Hz-200 Hz, 75 Hz-150 Hz, 75 Hz-125 Hz, 75 Hz-120 Hz, 75 Hz-115 Hz, 75 Hz-110 Hz, or 75 Hz-100 Hz. The amplitude of current may be 0.1 mA-30 mA, such as, 0.1 mA-25 mA, such as, 0.1 mA-20 mA, 0.1 mA-15 mA, 0.1 mA-10 mA, 1 mA-20 mA, 1 mA-10 mA, 2 mA-30 mA, 2 mA-15 mA, or 2 mA-10 mA.

The electrical stimulation may be applied in pulses such as a uniphasic or a biphasic pulse. The time span of a single pulse is referred to as the pulse width or pulse duration. The pulse width used in the present methods may vary widely depending on the numerous factors (e.g., severity of the disease, status of the patient, and the like) and may be determined empirically or may be pre-defined. In certain embodiments, the method may involve applying an electrical stimulation at a pulse width of about 10 μsec-990 μsec, for example, 30 μsec-990 μsec, 50 μsec-990 μsec, 75 μsec-990 μsec, 100 μsec-990 μsec, 200 μsec-990 μsec, 300 μsec-990 μsec, 500 μsec-990 μsec, 500 μsec-900 μsec, 30 μsec-900 μsec, 50 μsec-900 μsec, 75 μsec-900 μsec, 100 μsec-900 μsec, 200 μsec-900 μsec, 300 μsec-900 μsec, 500 μsec-900 μsec, 30 μsec-500 μsec, 50 μsec-450 μsec, 75 μsec-300 μsec, 100 μsec-200 μsec, or 100 μsec-550 μsec.

The electrical stimulation may be applied for a stimulation period of 0.1 sec-1 month, with periods of rest (i.e., no electrical stimulation) possible in between. In certain cases, the period of electrical stimulation may be 0.1 sec-1 week, 1 sec-1 day, 10 sec-12 hrs, 1 min-6 hours, 10 min-1 hour, and so forth. In certain cases, the period of electrical stimulation may be 1 sec-1 min, 1 sec-30 sec, 1 sec-15 sec, 1 sec-10 sec, 1 sec-5 sec, 1 sec-3 sec, 1 sec-2 sec, or 5 sec-10 sec. The period of rest in between each stimulation period may be 60 sec or less, 30 sec or less, 20 sec or less, or 10 sec. The amplitude of current may be 0.1 mA-30 mA, such as, 0.1 mA-25 mA, such as, 0.1 mA-20 mA, 0.1 mA-15 mA, 0.1 mA-10 mA, 1 mA-20 mA, 1 mA-10 mA, 2 mA-30 mA, 2 mA-15 mA, or 2 mA-10 mA.

The electrical stimulation having the parameters as set forth above may be applied over a program duration of around 1 day or less, such as, 18 hours, 6 hours, 3 hours, 1 hour, 45 minutes, 30 minutes, 20 minutes, 10 minutes, or 5 minutes, or less, e.g., 1 minute-5 minutes, 2 minutes-10 minutes, 2 minutes-20 minutes, 2 minutes-30 minutes, 5 minutes-10 minutes, 5 minutes-30 minutes, or 5 minutes-15 minutes which period would include the application of pulses and the intervening rest period. The program may be repeated at a desired program frequency until treatment of the subject is achieved. As such, a treatment regimen may include a program for electrical stimulation at a desired program frequency and program duration. In some embodiments, the treatment regimen may be controlled by a user via a pulse generator connected to the one or more stimulation electrodes in an open-loop treatment regimen. In other embodiments, the treatment regimen may be controlled by a control unit in communication with a pulse generator connected to the one or more stimulation electrodes in a closed-loop treatment regimen. Closed-loop treatment methods and systems are further described elsewhere in this disclosure.

As noted above, the treatment may ameliorate one or more symptoms of a disorder of dissociation or association suffered by the subject. By “therapeutically effective dose or amount” of electrical stimulation that modulates rhythmic neural activity in the posteromedial cortex or posteromedial cortical activity is intended an amount that, when the electrical stimulation is administered, as described herein, brings about a positive therapeutic response in the treatment of a disorder of dissociation or association. For example, a positive therapeutic response in the treatment of a disorder of dissociation may include a reduction in symptoms of dissociation, such as associated with a seizure or a dissociation-linked neuropsychiatric disorder such as post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder. A positive therapeutic response in the treatment of a disorder of association may include a reduction in symptoms of association, such as specific, recurrent, and/or negative thoughts that cause morbidity or mortality, associated with chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction/dependency, or other dysfunctional condition, wherein disconnecting from the damaging thoughts associated with the disorder is beneficial. The exact amount required will vary from subject to subject, depending on the species, age, and general condition of the subject, the severity of the condition being treated, the particular mode of administration, and the like. An appropriate “effective” amount in any individual case may be determined by one of ordinary skill in the art using routine experimentation, based upon the information provided herein.

The electrical stimulation may be delivered rhythmically or continuously during a stimulation period depending on the disorder being treated. For example, the electrical stimulation may be delivered rhythmically to induce dissociation for treatment of a disorder of association. Electrical stimulation may be delivered continuously to suppress rhythmic neural activity in the posteromedial cortex for treatment of a disorder of dissociation.

Assessment of effectiveness of treatment and assessment of amelioration of a symptom of a disorder of dissociation or association may be performed at any suitable time point after commencement of the treatment procedure, for example, after a treatment regimen is complete. Embodiments of the subject methods include assessing effectiveness of treatment or amelioration of a symptom of a disorder of dissociation or association within seconds, minutes, hours, or days after the initial treatment regimen has been completed. In some instances, assessment may be performed at multiple time points. In some cases, more than one type of assessment may be performed at the different time points. In some embodiments, a subject's brain activity (e.g., at one or more regions in the in the posteromedial cortex) may be measured prior to the application of electrical stimulation to the in the posteromedial cortex and assessing may include comparing the rhythmic neural activity in the posteromedial cortex after the treatment to that before the treatment, and a change in the post-treatment brain activity (e.g., suppressing or inducing rhythmic neural activity in the posteromedial cortex) may indicate successful treatment.

As such, the methods and systems provided herein may be used to ameliorate one or more symptoms of a disorder of dissociation or association in a patient. A person skilled in the art will appreciate that amelioration of a symptom may provide relief to a patient suffering from two separate neuropsychiatric conditions which share a symptom.

Upon completion of a treatment regimen, the patient may be assessed for effectiveness of the treatment and the treatment regimen may be repeated, if needed. In certain cases, the treatment regimen may be altered before repeating. For example, one or more of the frequency, pulse width, current amplitude, period of electrical stimulation, program duration, and/or program frequency may be altered before starting a second treatment regimen.

In certain cases, the treatment regimen may be tailored to the desired outcome. For example, for a patient suffering from an acute form of a disorder of dissociation or association, the treatment regimen may be chosen to provide an acute alleviation of one or more symptoms of the disorder. In contrast, the treatment regimen for a patient suffering chronically from a disorder of dissociation or association may be tailored for a chronic relief of one or more symptoms of the disorder. A treatment regimen for chronic relief of one or more symptoms of the disorder may also delay reappearance of one or more symptoms of the disorder and may even prevent the symptoms from appearing. In certain cases, a treatment regimen that is a combination of treatment regimens for acute and chronic alleviation of one or more symptoms of the disorder may be employed.

Application of the method may include a prior step of selecting a patient for treatment based on need as determined by clinical assessment, which may include cognitive assessment, anatomical assessment, behavioral assessment and/or neurophysiological assessment. In certain cases, a subject may be selected for treatment if the subject has epilepsy and is at risk of suffering from a seizure and dissociative symptoms. In certain cases, a subject may be selected for treatment if the subject has a dissociation-linked neuropsychiatric disorder such as, but not limited to, post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder, and is at risk of suffering from dissociative symptoms. In certain cases, a subject may be selected for treatment if the subject has condition or disease associated with producing an undesirable state of association in the subject. In certain cases, a subject may be selected for treatment if the subject has a disorder of association such as, but not limited to, chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction/dependency, or any dysfunctional association with specific, recurrent, and/or negative thoughts causing morbidity or mortality, wherein disconnecting from the damaging thoughts by generating a dissociative state is beneficial. Such a patient may also be implanted with intracranial stimulation electrodes at the posteromedial cortex region of the brain.

In certain aspects, the methods and systems of the present disclosure may include measurement of brain activity, for example, rhythmic neural activity in the posteromedial cortex. In certain cases, electrical activity from a plurality of locations in the posteromedial cortex may be measured and averaged. For example, electrical activity in the posterior cingulate, precuneus, and/or retrosplenial region may be measured. In certain cases, electrical activity may also be measured in the thalmus. In some cases, electrical activity in one or more locations in the brain may be measured during a period extending from prior to stimulation to the period during which stimulation to the brain is applied, or to a period after stimulation to the brain has been applied, and monitored for an increase or decrease in the rhythmic neural activity in the posteromedial cortex. In some cases, the application of electrical stimulation to the brain may increase or suppress rhythmic neural activity in the posteromedial cortex. In certain cases, the application of electrical stimulation to the brain may increase or suppress rhythmic neural activity in the posterior cingulate, precuneus, or retrosplenial region. In certain cases, the application of electrical stimulation to the brain may alter other neural features from one more regions of the brain. The alterations may be compared to the state of these features prior to the application of stimulation.

The foregoing methods may be performed as an open-loop or closed-loop method. For example, open loop or closed loop modulation could be used to prevent future dissociative episodes by constraining neural activity patterns (e.g., to suppress rhythmic neural activity in the posteromedial cortex). Open loop modulation could be used to alleviate acute symptoms of dissociation, triggered by neural activity (e.g., oscillations) or experienced symptoms by the patient. Open loop modulation could also be used to induce dissociation (e.g., to induce rhythmic neural activity in the posteromedial cortex) to alleviate acute symptoms of a disorder of association, for example, to manage traumatic situations, anxiety, panic, depression, chronic pain, obsessive-compulsive disorder, addiction, or dependency. In certain scenarios, it may be beneficial to induce dissociation without use of drugs.

Closed-loop methods and systems allow for automation of treatment of the subject including real-time need-based modulation of the treatment regimen. A closed-loop method allows determination of parameters of electrical stimulation based upon real-time feedback signals from the brain of the subject. In certain aspects, the subject methods operate as a closed-loop control system which may automatically adjust one or more parameters in response to electrical activity from a region of the brain of a subject. In some aspects, the closed loop system may be used to sense a subject's need for treatment using the methods disclosed herein. For example, the closed loop system may be programmed to monitor brain activity from one or more regions of the brain and compare the brain activity to a range indicative of a disorder. For treatment of dissociation in a patient with a dissociative disorder (e.g., epilepsy or dissociation-linked neuropsychiatric disorder), upon detection of electrical activity such as rhythmic neural activity in the posteromedial cortex indicative of the disorder, the closed loop system may automatically commence a treatment protocol of applying electrical stimulation.

Examples of Non-Limiting Aspects of the Disclosure

Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1-61 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below:

1. A method of inhibiting development of a dissociative state in a subject, the method comprising inhibiting a retrosplenial cortex layer-5 neuron optogenetically, wherein rhythmic neural activity is inhibited in the posteromedial cortex of the subject.

2. The method of aspect 1, wherein said inhibiting the retrosplenial cortex layer-5 neuron optogenetically comprises:

-   -   introducing a recombinant polynucleotide encoding a         light-responsive ion channel into the retrosplenial cortex         layer-5 neuron, wherein the light-responsive ion channel is         expressed in the retrosplenial cortex layer-5 neuron; and     -   illuminating the light-responsive ion channel with light at a         wavelength that activates the light-responsive ion channel,         wherein conduction of ions by the light-responsive ion channel         in response to absorption of light results in hyperpolarization         and inhibition of the retrosplenial cortex layer-5 neuron.

3. The method of aspect 2, wherein the light-responsive ion channel is a light-responsive anion-conducting opsin or a light-responsive proton conductance regulator.

4. The method of aspect 3, wherein the light-responsive anion-conducting opsin conducts chloride ions (Cl⁻).

5. The method of aspect 3 or 4, wherein the anion-conduction opsin is an anion-conducting channelrhodopsin or halorhodopsin.

6. The method of aspect 5, wherein the halorhodopsin is a Natronomonas pharaonis halorhodopsin (NpHR), enhanced NpHR (eNpHR) 1.0, eNpHR 2.0, or eNpHR 3.0.

7. The method of aspect 5, wherein the anion-conducting channelrhodopsin is iC1C2, SwiChR, SwiChR++, or iC++.

8. The method of aspect 3, wherein the light-responsive proton conductance regulator is a bacteriorhodopsin or an archaerhodopsin.

9. The method of aspect 8, wherein the light-responsive proton conductance regulator is Arch from Halorubrum sodomense, ArchT from Halorubrum sp., TP009 from Leptosphaeria maculans, or Mac from Leptosphaeria maculans.

10. The method of any one of aspects 2 to 9, wherein the polynucleotide encoding the light-responsive ion channel is provided by a viral vector.

11. The method of aspect 10, wherein the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector.

12. The method of aspect 10 or 11, wherein the viral vector is stereotactically injected into the retrosplenial cortex.

13. The method of any one of aspects 10 to 12, wherein the vector further comprises a neuron-specific promoter operably linked to the polynucleotide encoding the light-responsive ion channel.

14. The method of any one of aspects 10 to 13, wherein expression of the light-responsive ion channel is inducible.

15. The method of any one of aspects 1 to 14, wherein said illuminating the light-responsive ion channel comprises delivering light from a light source to the light-responsive ion channel using a fiber-optic-based optical neural interface.

16. The method of aspect 15, wherein the light source is a solid-state diode laser.

17. The method of any one of aspects 1 to 16, wherein the dissociative disorder is caused by a drug, a seizure, childhood psychological or physical trauma, abuse, sexual assault, or a dissociation-linked neuropsychiatric disorder.

18. The method of aspect 17, wherein the dissociation-linked neuropsychiatric disorder is post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder.

19. A method of inducing dissociative symptoms in a subject, the method comprising performing rhythmic optogenetic activation of a retrosplenial cortex layer-5 neuron, wherein rhythmic neural activity is induced in the posteromedial cortex of the subject.

20. The method of aspect 19, wherein said performing rhythmic optogenetic activation of a retrosplenial cortex layer-5 neuron comprises:

-   -   introducing a recombinant polynucleotide encoding a         light-responsive ion channel into the retrosplenial cortex         layer-5 neuron, wherein the light-responsive ion channel is         expressed in the retrosplenial cortex layer-5 neuron; and     -   rhythmically illuminating the light-responsive ion channel with         light at a wavelength that activates the light-responsive ion         channel, wherein conduction of ions by the light-responsive ion         channel in response to absorption of light results in         depolarization and activation of the retrosplenial cortex         layer-5 neuron.

21. The method of aspect 20, wherein the light-responsive ion channel is a light-responsive cation-conducting opsin.

22. The method of aspect 21, wherein the light-responsive cation-conducting opsin conducts calcium cations (Ca²⁺).

23. The method of aspect 21 or 22, wherein the light-responsive cation-conducting opsin is a light-responsive cation-conducting channelrhodopsin.

24. The method of aspect 23, wherein the light-responsive cation-conducting channelrhodopsin is a Chlamydomonas reinhardtii channelrhodopsin or a Volvox carteri channelrhodopsin.

25. The method of aspect 24, wherein the light-responsive cation-conducting channelrhodopsin is a Chlamydomonas reinhardtii channelrhodopsin-1 (ChR1), a Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2), a Volvox carteri channelrhodopsin-1 (VChR1), or a chimeric ChR1-VChR1 channelrhodopsin.

26. The method of any one of aspects 20 to 25, wherein the polynucleotide encoding the light-responsive ion channel is provided by a viral vector.

27. The method of aspect 26, wherein the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector.

28. The method of aspect 26 or 27, wherein the viral vector is stereotactically injected into the retrosplenial cortex.

29. The method of any one of aspects 26 to 28, wherein the vector further comprises a neuron-specific promoter operably linked to the polynucleotide encoding the light-responsive ion channel.

30. The method of any one of aspect 26 to 29, wherein expression of the light-responsive ion channel is inducible.

31. The method of any one of aspects 20 to 30, wherein said rhythmically illuminating the light-responsive ion channel comprises delivering light from a light source to the light-responsive ion channel using a fiber-optic-based optical neural interface.

32. The method of aspect 31, wherein the light source is a solid-state diode laser.

33. The method of any one of aspects 20 to 32, wherein said rhythmically illuminating the light-responsive ion channel with light comprises repeatedly delivering light at the wavelength that activates the light-responsive ion channel with a frequency of 2 Hz.

34. The method of any one of aspects 20 to 33, wherein said rhythmically illuminating the light-responsive ion channel with light comprises alternating between a stimulation interval and a non-stimulation interval, wherein the light at the wavelength that activates the light-responsive ion channel is delivered during the stimulation interval, and no light or light that is not at the wavelength that activates the light-responsive ion channel or that inhibits the light-responsive ion channel is delivered during the non-stimulation interval.

35. The method of aspect 34, wherein the stimulation interval is 100 milliseconds to 300 milliseconds.

36. The method of aspect 35, wherein the stimulation interval is 250 milliseconds.

37. The method of any one of aspects 34 to 36, wherein the non-stimulation interval is 100 milliseconds to 300 milliseconds.

38. The method of any one of aspects 19 to 37, wherein the subject has a disorder of association.

39. The method of aspect 38, wherein the disorder of association is chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction, or dependency.

40. A method of inducing dissociative symptoms in a subject, the method comprising: positioning an electrode to deliver electrical stimulation to a posteromedial cortex region of the brain of the subject; and applying electrical stimulation to the posteromedial cortex region using the electrode in a manner effective to generate rhythmic neural activity in the posteromedial cortex of the subject.

41. The method of aspect 40, wherein the method comprises applying electrical stimulation to a right posteromedial cortex region or a left posteromedial cortex region.

42. The method of aspect 41, wherein the electrode is placed on a surface of the right posteromedial cortex region or the left posteromedial cortex region.

43. The method of any one of aspect 41, wherein the electrode is placed within the right posteromedial cortex region or the left posteromedial cortex region.

44. The method of any one of aspects 40 to 43, wherein said applying electrical stimulation comprises delivering electrical stimulation to a posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the subject.

45. The method of any one of aspects 40 to 44, wherein the electrode is a non-brain penetrating surface electrode array or a brain-penetrating electrode array.

46. The method of any one of aspects 40 to 45, wherein the subject has a disorder of association.

47. The method of aspect 46, wherein the disorder of association is chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction, or dependency.

48. The method of any one of aspects 40 to 47, wherein said electrical stimulation is applied at 2 mA to 10 mA.

49. The method of any one of aspects 40 to 48, wherein said electrical stimulation is applied for up to 2 seconds.

50. The method of aspect 49, wherein said electrical stimulation is applied for 1 second to 2 seconds.

51. The method of any one of aspects 40 to 50, wherein the electrical stimulation is applied unilaterally or bilaterally.

52. The method of any one of aspects 40 to 51, wherein the electrical stimulation is applied with a frequency of 50 Hz.

53. The method of any one of aspects 40 to 52, further comprising mapping the brain of the subject to identify an optimal location in the posteromedial cortex for positioning the electrode for delivery of the electrical stimulation to induce dissociative symptoms in the subject.

54. The method of any one of aspects 52 to 55, wherein the electrical stimulation is delivered rhythmically.

55. A method of modulating associative or dissociative symptoms in a subject, the method comprising delivering electrical stimulation to a posteromedial cortex region of the brain of the subject that modulates rhythmic neural activity in the posteromedial cortex of the subject.

56. The method of aspect 55, wherein the electrical stimulation is delivered optogenetically, with an electrode, or by transcranial magnetic stimulation.

57. The method of any one of aspect 56, wherein the electrode is a non-brain penetrating surface electrode array or a brain-penetrating electrode array.

58. The method of aspect 55 to 57, wherein the electrical stimulation is delivered to a posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the subject.

59. The method of any one of aspects 55 to 58, wherein the electrical stimulation is delivered rhythmically or continuously during a stimulation period.

60. The method of any one of aspects 55 to 59, further comprising mapping the brain of the subject to identify an optimal location in the posteromedial cortex for positioning the electrode for delivery of the electrical stimulation to modulate associative or dissociative symptoms in the subject.

61. The method of any one of aspects 55 to 60, wherein the electrical stimulation is applied unilaterally or bilaterally.

Experimental

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference.

The present invention has been described in terms of particular embodiments found or proposed by the present inventor to comprise preferred modes for the practice of the invention. It will be appreciated by those of skill in the art that, in light of the present disclosure, numerous modifications and changes can be made in the particular embodiments exemplified without departing from the intended scope of the invention. All such modifications are intended to be included within the scope of the appended claims.

Example 1 Deep Posteromedial Cortical Rhythm Underlying Dissociation

Here we investigate the dissociative-like behavioral state with high-speed brain-wide approaches in both mouse and human, identifying underlying deep posteromedial-cortex rhythmic dynamics along with molecular, cellular, and physiological mechanisms.

Advanced imaging methods allow cell type-specific recording of neural activity across the mammalian brain, enabling exploration of how brain-wide dynamical patterns give rise to complex behavioral states¹⁻¹². Dissociation is an altered behavioral state with unknown underlying neurophysiology but substantial basic and clinical significance—that can occur with trauma, epilepsy, or dissociative drug use^(13,14)—in which the integrity of experience is disrupted, and reproducible cognitive phenomena result (including dissociation of stimulus detection from stimulus-related affective responses). Here we have established such a dissociation-like state in mice, using precisely-dosed ketamine or phencyclidine. Large-scale imaging of neural activity revealed that these dissociative agents elicited a 1-3 Hz rhythm in retrosplenial cortex layer-5 neurons. Electrophysiological recording with four simultaneously-deployed high-density probes revealed rhythmic coupling of retrosplenial cortex and anatomically-connected anterior thalamus circuitry, but uncoupling from most other brain regions, including an inverse correlation with frontally-projecting thalamic nuclei. In testing for causal significance, rhythmic optogenetic activation of retrosplenial cortex layer-5 neurons recapitulated the dissociation-like behavioral effects. Local retrosplenial HCN1 pacemaker channels were required for systemic ketamine to induce this rhythm and to elicit dissociation-like behavioral effects. In a patient with focal epilepsy, simultaneous intracranial stereo-EEG recordings from across the brain revealed a similarly-localized rhythm in the homologous deep posteromedial cortex temporally correlated with pre-seizure self-reported dissociation, and local brief electrical stimulation elicited dissociative experiences. These results identify molecular, cellular, and physiological properties of a conserved deep posteromedial cortical rhythm underlying states of dissociation.

Results Multiregional Imaging Reveals Ketamine-Induced Rhythm Restricted to Retrosplenial Cortex

We recorded multiregional neuronal activity by leveraging widefield microscopy, a cleared skull preparation, and atlas registration in GCaMP6s-expressing mice (FIGS. 1A-1C)^(6,8,15). Upon intraperitoneal injection of ketamine (50 mg/kg subanesthetic dose; Methods), a 1-3 Hz oscillation emerged in retrosplenial cortex (RSP), but not in any other cortical region (FIGS. 1D-1 F, FIG. 6 a ; n=5 mice). The rhythm emerged within 120 seconds, returned to baseline after ˜45 minutes (FIG. 1G, Extended Data FIGS. 1B-1 E), and exhibited similar magnitude across five consecutive days; no ketamine-induced 1-3 Hz movements were observed (FIGS. 6G-6L). Subanesthetic ketamine reduced RSP activity-correlations with all other dorsal-cortical regions (FIG. 6J). Using high-magnification two-photon microscopy we imaged superficial neuropil through a glass-covered cranial window, and observed the oscillation in RSP but not neighboring visual cortex (FIGS. 6K-6O; n=4 mice, paired t-test p=0.027).

In humans, ketamine can cause dissociation, analgesia, hallucinations, sedation, and anesthesia. To test whether other drugs with or without dissociative properties elicited similar cortical activity patterns, we repeated imaging with NMDAR-antagonists, a hallucinogen, anesthetics, a sedative, and an analgesic. PCP, a dissociative NMDAR-antagonist like ketamine, induced the RSP-localized oscillation (FIG. 1H), as did MK-801, a long-lasting dissociative NMDAR-antagonist. Memantine, a low-affinity uncompetitive NMDAR-antagonist with a non-dissociative clinical profile did not elicit oscillation, nor did saline or lysergic acid diethylamide (LSD) (FIG. 7 ). Non-dissociative anesthetics dexmedetomidine, propofol and the commonly-used veterinary ketamine/xylazine cocktail, elicited waves of cortical activity without RSP restriction (FIG. 8 ). Neither the GABAergic sedative diazepam nor the centrally-acting opioid-analgesic buprenorphine elicited the rhythm (FIG. 9 ). Together, these data revealed an oscillatory pattern spatially restricted to RSP selectively induced by dissociative agents.

Retrosplenial Oscillation Localized to Layer 5 Neurons

This multi-regional perspective revealed spatially-localized dynamics but did not enable observation of layer-specific cellular contributions. We therefore restricted GCaMP6m expression to specific cortical layers and measured single-neuron RSP Ca²⁺ signals (Extended Data FIGS. 5A 5B). In layer 2/3 neurons (Cux2-CreER mice¹⁶), neither oscillating neurons nor population synchrony were observed (FIG. 1I, FIGS. 100, 10D n=5 animals, paired t-test, p=0.7). In contrast, layer 5 neurons (Rbp4-Cre mice¹⁷) exhibited synchronous activity (FIGS. 1I, 10E, 10F). We verified layer-specificity with brain-wide activity-mapping using the TRAP2 mouse line^(18,19); layer 5 was specifically recruited (unpaired t-test, p<0.001) (FIG. 1J, FIGS. 10G-101 ). Thus, under these conditions, ketamine elicited rhythmicity selectively in layer 5 RSP neurons.

Synchronized Spiking in Deep RSP and Subcortical Brain Areas

We extended these findings to the single-spike millisecond-scale domain, electrophysiologically recording with 32-channel two-shank silicon probes. In 50 mg/kg ketamine-injected animals, deep RSP neurons engaged in intermittent ˜250 ms synchronous bursts, separated by ˜250 ms silent states (FIG. 2A, FIGS. 10J, 10K). Individual units fired ˜5-10 spikes/burst; nearly all identified units participated in every burst event, and most bursts contained nearly all units, with structured temporal ordering (FIGS. 10L-10P).

We constructed a four-probe long-shank high-contact-density Neuropixels recording system to simultaneously record hundreds of neurons throughout the brain (FIGS. 2B, 2C, FIG. 11A). Subanesthetic ketamine influenced several brain regions (FIG. 2D, FIGS. 11B, 11C). As expected, we observed the 1-3 Hz RSP rhythm—but also discovered specific subcortical regions (inaccessible to cortical imaging) exhibiting increased rhythmicity (FIGS. 11D-11G). Whereas pre-ketamine nearly-all brain regions were correlated with RSP, 8-12 min after ketamine injection correlations with RSP were reduced (notably in somatosensory cortex, subiculum, ventral/anteromedial thalamus, and the red nucleus (FIGS. 2E, 2F, FIGS. 12A-12E, FIG. 13 ).

A surprising uncoupling was observed between adjacent (but differently-connected) thalamic nuclei. Whereas laterodorsal and anteroventral thalamus (with known posterior cortical wiring relationships including to RSP²⁰⁻²²) became more-strongly correlated to each other and RSP, anteromedial thalamic neurons (with known projections to frontal cortex²⁰⁻²²) were found to oscillate out-of-phase with RSP (FIGS. 2F, 2G, FIGS. 12F-121 ). Thus, brain-wide electrophysiology under subanesthetic ketamine revealed a globally-detectable disconnection motif, interpretable via the rhythm and wiring of RSP.

Dissociative-Like Behavioral Effects Tracking RSP Rhythm

Mice were behaviorally-tested extensively, beginning with reflexive (paw-flick), affective/self-protective (paw-licking) and motivational (jump-to-escape) responses to aversive stimuli (hot-plate test; FIG. 3A). While ketamine did not reduce reflexive responses (paw-flick, one-way ANOVA, F_(4,20)=1.11, p=0.38), revealing robustly-preserved sensory-detection and motor capabilities, ketamine abolished affective/emotional (paw-lick, one-way ANOVA F_(4,20)=10.1, p<5E-4) and motivational (jump-to-escape, one-way ANOVA F_(4,20)=105, p<1E-12) defensive behaviors at 25 and 50 mg/kg (FIG. 3B, FIG. 14A), with similar effects on rearing and behavioral-latency (FIGS. 14B, 14C).

This separability of stimulus-detection from affective-response suggested a dissociation-like state. We next explored longer-timescale escape and social interactions. Ketamine>=25 mg/kg similarly suppressed tail-suspension escape responses (one-way ANOVA, F_(4,20)=9.36, p=0.0002) and resident-intruder interactions (one-way ANOVA, F_(4,15)=13.6, p<0.0001) (FIG. 14 d,e ); consciousness was maintained as mice ambulated spontaneously and responded to external stimuli, and the righting-reflex test for consciousness was preserved in all animals (but abolished by anesthetic ketamine; 200 mg/kg, FIG. 14F).

Affective behaviors were consistently preserved at 13 mg/kg, yet selectively abolished at 25 mg/kg. We therefore directly compared RSP activity between these two doses using both widefield imaging, and fiber photometry with locally-expressed GCaMP6m for increased specificity. RSP recordings revealed a 1-3 Hz oscillation at 25 mg/kg—but not 13 mg/kg—suggesting that the 1-3 Hz RSP rhythm could be critical for dissociation-like behavioral effects (FIGS. 3C, 3D).

At dissociative doses, ketamine can also induce analgesia, hallucinations, and sedation; we therefore repeated the behavioral panel with four additional classes of drugs that elicit subsets of these effects. The non-sedative dissociative agent PCP alone recapitulated ketamine's behavioral disconnection between sensory-detection and affective responses in the hot-plate test; like ketamine, PCP also inhibited longer-timescale escape and social behaviors (FIG. 3E, FIG. 14G-14K); locomotion velocity was unchanged (FIG. 3F, FIG. 14L). Analgesics (centrally-acting buprenorphine and topical lidocaine) decreased stimulus-detection foot-flicks; neither diminished escape responses (FIG. 14L). The hallucinogen LSD disrupted neither sensory-detection nor affective responses, and tended to increase responsivity to salient stimuli (decreased time-to-jump, increased rearing/socialization) while also reducing locomotion in the stimulus-poor open-field. Finally, the sedative diazepam did not recapitulate dissociative-like behavior.

Together, these results revealed a measurable and consistent dissociation-like behavioral phenotype in mice specifically elicited by dissociative agents (preserved stimulus-detection with diminished affective response) at doses corresponding to emergence of the RSP rhythm.

Rhythmic Drive of RSP Layer 5 Neurons: Elicitation of Dissociation-Like Effects

To test causal impact of this oscillation, we optogenetically delivered the corresponding activity to RSP layer 5 neurons by co-expressing eNpHR3.0/ChR2 (eNPAC) in layer 5 (Rpb4+) RSP and rhythmically-stimulating at 2 Hz (250 ms/20 Hz blue light, alternating with 250 ms continuous yellow light) (FIG. 3G, FIG. 15A). Controls included non-opsin-expressing animals and non-rhythmic (random) illumination of opsin-expressing animals.

While rhythmic RSP drive did not disrupt reflexive responses, affective paw-licking was reduced vs. control (unpaired t-test, corrected p=0.03) (FIG. 3G). Rhythmic drive lengthened time-to-escape vs. non-opsin (unpaired t-test, corrected p=0.02) and random/non-rhythmically-illuminated animals (unpaired t-test, corrected p=0.05) (FIG. 15B), and rearing was reduced in rhythmically-illuminated animals vs. control (unpaired t-test, corrected p=0.02). Rhythmic illumination significantly diminished tail-suspension escape behavior (for which repeated within-animal testing was possible) compared with random illumination in eNPAC-expressing animals (paired t-test, rhythmic vs. random, p=0.01) (FIG. 15E). Rhythmic drive did not reduce resident-intruder social interactions (paired t-test, rhythmic vs. random, p=0.14; Extended Data 10F); righting-reflex and open-field behaviors were unaffected. To test rhythmic optogenetic drive of a different cortical area, we expressed eNPAC in deep layers of somatosensory cortex; here, rhythmic illumination did not induce the dissociative behavioral state (FIG. 3G, FIGS. 15L-15O). In summary, optogenetically providing the activity pattern naturally arising in layer 5 RSP neurons in response to ketamine exerted the effect of dissociating sensory and affective responses.

Physiological and Behavioral Effects of Ketamine Require RSP Rhythm-Generator

How could systemic ketamine evoke such a layer-specific, focal, and behaviorally-potent rhythm? We explored two major hypotheses: (1) long-range subcortical inputs provided a specific oscillatory drive to layer-5 retrosplenial neurons; or (2) selective expression of specific ion channels in retrosplenial cortex facilitated local oscillations (FIG. 4A).

Since thalamic nuclei have been implicated in rhythmicity²³⁻²⁶, and exhibited oscillations in our electrophysiology, thalamo-cortical loops might contribute to RSP rhythms. To test this hypothesis, we optogenetically inhibited monosynaptic-excitatory inputs (dorsal-thalamus or subiculum) while recording RSP activity after ketamine injection (FIGS. 4B, 4C, FIG. 16 a,b ). Inhibition of dorsal thalamus did not attenuate, but rather enhanced, the cortical oscillation (paired t-test, corrected p=0.003) (FIG. 4C, FIG. 16C); inhibition of subiculum (or illumination of control animals) had no effect.

To test local rhythm-generators, we investigated two candidate channels: NMDA-receptors (a primary target of ketamine^(27,28) and PCP) and HCN1 pacemaker channels (involved in spontaneous rhythmic firing^(29,30)). Immunohistochemistry of HCN1 channels, but not NMDA receptors, revealed high expression localized to deep RSP (relative to layer 2/3 RSP and neighboring deep visual cortex; FIG. 4D, FIGS. 16D, 16I). We recorded from RSP after NMDAR (GRIN1) or HCN1 channels were genetically-disrupted via local injection of AAVdj-Ef1a-Cre and AAVdj-Ef1a-DIO-GCaMP6m viruses into RSP of adult homozygous floxed-channel transgenics, such that GCaMP6m would only be expressed in Cre-expressing and gene-knockout cells (FIG. 4E, FIGS. 15E-15F). After four weeks, ketamine-induced oscillations were significantly diminished in HCN1 and GRIN1 mice, but preserved in wild-type (FIG. 4E, FIG. 16G). By leveraging local-knockout mice, we could thus induce a brain state in which all aspects of global ketamine action would be preserved except for those depending on channels in retrosplenial cortex (without directly inhibiting or lesioning RSP—thus permitting, for example, non-oscillatory RSP activity).

For behavioral experiments, mice were multiply-injected across RSP, yielding local reduction in protein expression (FIGS. 16H-16J). In controls, 50 mg/kg ketamine abolished affective paw-licking while preserving reflexive paw-flicks—the dissociative phenotype (FIG. 4F). RSP injection of Cre-virus in HCN1-KO mice strikingly restored affective paw-licking (Mann-Whitney U, corrected p<0.001); thus, ketamine-imposition of dissociation-like behavior was blocked. Both channel-knockouts also showed modestly-recovered tail suspension-escape behavior (p<0.001) and resident-intruder social interactions (p<0.01) vs. wild-type; all mice successfully-righted postural inversion (FIGS. 16K-16N). Without ketamine, AAVdj-Ef1a-Cre-injected HCN1-KO and GRIN1-KO animals exhibited reflexive and affective behaviors comparable to wild-type C57BL/6 mice (FIGS. 15P-15S). Thus, local RSP HCN1 pacemakers were required for systemic ketamine to induce the deep RSP rhythm and elicit the dissociation-like behavioral state.

Focal 3 Hz Rhythm in Human Deep Posteromedial Cortex Linked to Self-Reported Dissociation

An epilepsy patient at the Stanford Comprehensive Epilepsy Center (with pre-seizure auras described as dissociative) had been previously implanted with brain-wide intracranial electrodes (stereoelectroencephalography) for diagnostic recording/stimulation. Epileptologist inspection of sEEG traces near seizure-onset with dissociative aura revealed a prominent rhythmic waveform only in deep posteromedial cortex (PMC; defined as retrosplenial, posterior cingulate, and medio-ventral precuneus cortex; rodent RSP is considered the closest analog to human PMC areas³¹) (FIGS. 5A-5C; FIGS. 12A, 12B; FIG. 18 ). Applying the same analyses from mouse imaging, we discovered a sharp oscillatory profile centered on 3.4 Hz restricted to deep PMC (FIGS. 5D, 5E, FIG. 17C). Although the seizure focus was localized to the right hemisphere, left PMC engaged in simultaneous oscillatory dynamics, giving rise to bilaterality as in mouse.

As part of clinical mapping, brief stimulation (50 Hz, 2-10 mA, total duration 1.3+/−0.47 seconds; mean+/−s.e.m.) was applied at individual electrodes. To quantify the elicited response from each stimulation in unbiased fashion, we identified every electrical contact that exhibited rhythmicity during spontaneous dissociative auras (FIG. 17D). Stimulation of the seizure focus in right PMC elicited dissociative, aura-like feelings similar to seizure onset (FIG. 5F). Stimulation of left PMC elicited immediate and confidently-reported dissociative experiences without the negative valence of an impending seizure (comments 4-6). Stimulations through these spontaneously-oscillating PMC contact sites evoked a dissociative aura 11/13 times, whereas virtually no non-oscillating contacts responded in this way (FIG. 5F, FIG. 17E). Only one sham stimulation elicited report of an aura; this one report followed a real stimulation that had elicited a strong aura. Together, these results demonstrated causal elicitation of human dissociative symptoms local to sites exhibiting the deep PMC rhythm.

Discussion

Here, large-scale optical recording technology enabled discovery of a dissociative agent-elicited, layer 5-restricted, low-frequency rhythm localized to deep RSP. This rhythm was associated with elicitation of a dissociation-like behavioral state in mice, and precisely-paced optogenetic intervention revealed that the rhythmic activity itself was causally linked to the dissociative-like effects. RSP-localized knockout of HCN1 channels abolished both the pharmacologically-induced rhythm and dissociation-related behavior. Conscious experience of human dissociation was linked to endogenous rhythmic activity in the homologous deep PMC. These experiments identified molecular, cellular, and physiological properties of a deep posteromedial cortical rhythm underlying dissociation-like states.

Key aspects of dissociation were recapitulated by three specific interventions: administration of retrosplenial rhythm-inducing doses of ketamine or PCP in mice, rhythmic RSP-localized optogenetic stimulation in mice, and similarly-localized electrical stimulation in a patient with epilepsy originating from right PMC (FIG. 19 ). We note that high-frequency electrical stimulation of non-epileptic PMC (spanning superficial and deep regions across many human subjects, in the absence of the epileptic tissue-dependent low-frequency rhythm observed here) does not induce dissociation³², consistent with our result that low-frequency rhythmic optogenetic drive was specifically and causally linked to dissociative-like behavioral effects. High-frequency electrical intervention, when delivered to epileptic tissue with specific wiring or ion channel-expression properties, could cause dissociation by several possible cellular mechanisms (including induced rhythmic activity). Likewise ketamine may induce additional effects in human beings beyond RSP rhythm that contribute to its behavioral properties³³⁻³⁵.

Biophysical mechanisms of both the rhythm and the behavioral state were addressable in the mouse. HCN1 channels underlie I_(h), a hyperpolarization-activated depolarizing current that can pace rhythmic activity; we found that RSP-specific HCN1 disruption selectively abolished both the ketamine-induced rhythm and dissociation-like behavioral effects. Ketamine blockade of specific depolarizing channels (such as NMDARs) could allow RSP membrane potentials to hyperpolarize, activating intrinsic HCN1 channels and permitting rhythmic dynamics. Our unexpected observations regarding optogenetic inhibition of long-range inputs to RSP, which enhanced (rather than suppressed) ketamine-induced oscillations, were consistent with this model for HCN1 function in posteromedial cortical rhythmicity, as the long-range inputs are likely glutamatergic and excitatory. Cells with certain high HCN1 expression levels may be most susceptible to this effect, a prediction consistent at the regional level with the spatial pattern of cortical HCN1-expression observed and our TRAP2 activity-localization results. The reliable temporal ordering of single-unit spike sequence during each oscillation cycle observed from electrophysiology might reflect the impact of such specific ion channel-expression on membrane dynamics at the single-cell level; future work in humans will be important for probing further how specific differential expression of rhythm-generating channels may contribute to subjective disorders of dissociation. In GRIN1 knockouts, effects were less dramatic than for HCN1 knockouts, and the rhythm's magnitude was partially attenuated without recovery of affective behavior; remaining oscillatory dynamics could be of sufficient potency to cause dissociation, or alternatively the rhythm-attenuated brain state would be permissive for affective behavior expression if postsynaptic temporal integration via functional NMDA receptors were intact.

Regarding functional importance of the ketamine-elicited thalamic oscillation pattern, it is intriguing to consider that the de-coupling of ‘primary’ thalamic systems (LD, AV; known to connect robustly with posterior forebrain circuitry) and ‘secondary’ thalamic systems (including AM, projecting to frontal circuitry) could contribute to dissociative symptoms. The inverse correlation of AM from AV, LD, and RSP could affect autobiographical cognition and egocentricity by disconnecting frontal cortical areas from posterior areas, while the 1-4 Hz rhythmicity could maintain continuity of neural activity for >200 ms in each subnetwork to preserve conscious mental experience (distinguishably from anesthesia).

Of all the neocortical regions observed, only PMC exhibited rhythmic activity linked to both dissociative pharmacology and dissociative aura. Interestingly, prior work had suggested that effects of NMDAR-antagonizing agents could be greatest in RSP or PMC36,37, but the capability for independent functional operation of this cortical region (as revealed here) was surprising in light of data from many laboratories pointing to extensive cortico-cortical wiring and dynamics that integrate neocortex into a distributed network. Such findings largely come from task-performing animals, while human imaging data report PMC to be less active in externally-oriented tasks and more active during stimulus-independent thought, autobiographical cognition, and mind wandering—all self-oriented, task-independent psychological states^(38,39).

Future clinical work may include exploring whether electrically-induced low-frequency rhythms in healthy human deep PMC can cause dissociative states, and whether dissociative drugs (or dissociation-linked neuropsychiatric states including PTSD and borderline personality disorder) involve these rhythms in humans. Dissociation in human beings is a self-reported state of mind, and no experimental technique in rodents or humans can currently define in full this subjective experience. However, the clinically-guided paradigm reported here may provide a framework of behavioral, optical, and physiological tools enabling exploration of neural activity underlying dissociative states. These results highlight the value of new brain-scale recording technologies; here, initial optical and electrical multiregional activity-screening guided further quantitative testing of precise causal hypotheses. More generally, integrative technologies with broad and high-resolution perspective may provide increasingly-informative experimental access to internal representations of sensations, cognitions, and actions at cellular resolution (and with region-wide or even brain-wide perspective), providing a path forward for elucidating the dynamics involved in creating complex brain and behavioral states important in health and disease.

Methods Experimental Model and Subject Details

All procedures were in accordance with protocols approved by the Stanford University Institutional Animal Care and Use Committee (IACUC) and guidelines of the National Institutes of Health. Mouse strains used were C57BL/6J (Black 6, Jackson Laboratory, #664), Tg(Thy1-GCaMP6s)GP4.3Dkim (Thy1-GCaMP6s, JAX #024275), Cux2-CreERT2 (gift of S. Franco, University of Colorado), Ai148(TIT2L-GC6f-ICL-tTA2-D (Ai148, Jax #030328) (gift of H. Zeng, Allen Institute for Brain Science), Tg(Rbp4-cre)KL100Gsat (Rbp4-Cre, No. 031125-UCD, MMRRC) (gift of L. Luo, Stanford), B6.129S-HcnitmlKndl/J (HCNV Jax #028299) (gift of L. Giocomo, Stanford), B6.129S4-Grin1tm2StI/J (NR1vf Jax #005246). Male and female mice were used, aged 6-24 weeks at the time of surgery. Mice were group housed in plastic cages with disposable bedding on a standard light cycle until surgery and behavior, when they were moved to a 12 hr reversed light cycle.

Drug Administration

Drugs used were ketamine (VEDCO, ketamine HC1, 6-200 mg/kg), phencyclidine (PCP, 5 mg/kg, Sigma-Aldrich P3029), memantine (50 mg/kg), MK801 (0.75-1 mg/kg), dexmedetomidine (0.35-1.5 mg/kg), propofol (35-140 mg/kg), xylazine (AnaSed AKORN, 12.5 mg/kg), buprenorphine SR (2 mg/kg), lidocaine (topical), LSD ((+)-Lysergic acid diethylamide (+)-tartrate (2:1), 0.3 mg/kg), and diazepam (2 mg/kg). For all imaging, recording, and behavioral experiments, drugs were injected intraperitoneally (with a 26-gauge needle) 10 minutes before data was taken unless noted otherwise (e.g. see ketamine injection for Neuropixels experiment). For injection during imaging or Neuropixels recording, a catheter was implanted intraperitoneally before head-fixation (BD Saf-T-Intima #383323). For anti-depressant effects, human doses are typically 0.3-1 mg/kg, and in mouse 3-10 mg/kg. Following the same pattern, humans consistently report potent dissociation at ˜2-3 mg/kg, and in the mouse, the lowest tested dose causing the oscillation and behavioral effects was 25 mg/kg⁴⁰⁻⁴². Thus, the dose-response relationship for dissociative effects of ketamine in mice corresponds with human/mouse mapping for previously-studied effects of ketamine.

Pan-Cortical Imaging with Blood Autofluorescence Correction

As described⁸, mice were anesthetized with isoflurane, the scalp was removed, the skull cleaned and dried, and a custom head-plate was cemented to contacts over the cerebellum and in front of the olfactory bulb. The skull was then covered in a thin layer of cyanoacrylate glue (Apollo 2000, Cyberbond), clear dental acrylic (Ortho-Jet, Lang Dental), and clear nail polish (Electron Microscopy Services). Buprenorphine SR (0.1 mg/kg) was injected subcutaneously for pain management. Mice were given >=1 wk recovery before experiments. Imaging was performed on a custom-built fluorescence microscope designed for high light collection efficiency and large field of view. The microscope consisted of back-to-back 50 mm f/1.2 camera lenses (Nikon), separated by a FF495-D103-50.8-D dichroic mirror (Semrock), mounted in a 60 mm cube (Thorlabs). An F-mounted ORCA Flash 4.0 (Hamamatsu) was used to record images, with a FF01-520/35-50.8-D emission filter (Semrock). Alternating 410 nm and 488 nm illumination for non-Ca²⁺ dependent artifact removal was controlled using a microcontroller (Arduino) slaved to the frame output trigger of the camera.

Two-Photon Imaging

Mice were anesthetized with isoflurane and the skull above retrosplenial cortex was removed and replaced with a 7 mm circular glass coverslip. For layer 1 experiments, ThylGcaMP6s mice were used. For layer 2/3 experiments, Cux2-CreER;Ai148 mice were used (tamoxifen dissolved in corn oil was injected intraperitoneally at 0.1 mg/g at least 2 weeks prior to imaging). For layer 5 experiments, Rbp4-Cre mice were injected locally in retrosplenial with 800 nL AAVdj-Ef1a-DIO-GCaMP6f (titer 5E12) at stereotactic coordinates (−3AP, 0.5 ML, −0.6DV). Mice were given >=1 week to recover before imaging. Awake, head-fixed imaging was performed on a standard two photon microscope (NeuroLabWare, Los Angeles, CA). A 16×0.8 NA Nikon objective was immersed in clear ultrasound gel for imaging (Aquasonic, Parker Laboratories); frame rate of 30 Hz. Although we observed oscillations in RSP with 50 mg/kg, we were concerned that 50 mg/kg may be too low a dose to sensitively detect a potentially weaker signal in visual cortex or layer 2/3, and so we used 80 mg/kg in these experiments.

Ketamine-TRAP Experiments

Dissolved 4-OHT (20 mg/mL in ethanol) was mixed with corn oil at a concentration of 10 mg/mL, and ethanol evaporated by vacuum centrifugation (60 minutes). Mice were each placed alone in a novel cage for 30 minutes, to remain for the experiment. Two mice were injected with 50 mg/kg ketamine intraperitoneally, and two mice were injected with an equivalent volume of saline. After 1 hr, all mice were injected with 50 mg/kg tamoxifen, as well as an additional dose of 50 mg/kg ketamine/saline. After 1 hr, a final dose of ketamine/saline was administered. This protocol was designed to ensure that the ketamine-induced oscillation (and associated cFos expression) was occurring throughout the majority of the ˜4 hr post-tamoxifen-injection time window during which TRAP2 labels cells. After 10 days (to allow time for expression of tdTomato), mice were perfused with ice cold PBS and 4% PFA. Coronal sections (50 μm) were taken using a vibratome and imaged on an epifluorescence microscope.

In Vivo Electrophysiology Retrosplenial Targeted Recordings

Mice were anesthetized with isoflurane, a small craniotomy (˜2 mm diameter) was performed above retrosplenial cortex, and a custom head-plate implanted. The dura was gently removed, and then covered with Kwik-Cast Sealant (World Precision Instruments). A skull screw was implanted and secured with cement in the right frontal skull bone. Mice were given 1-2 hours to recover. Next, mice were i.p. injected with 50 mg/kg ketamine, and head-fixed under an in vivo electrophysiology recording apparatus. To record neural activity, a 32-channel two-shank silicon neural probe (ASSY-37 P-1, Cambridge NeuroTech) was slowly lowered into retrosplenial cortex. The deepest tip of the electrode was 800-1000 μm from the brain surface for all recordings.

Simultaneous Recording from Four Neuropixels Probes with i.p. Drug Delivery

Surgeries

Mice were anesthetized with isoflurane, carefully leveled in a stereotaxic apparatus and their scalp and periosteum were removed. A 3% hydrogen peroxide in saline solution was used to clean the surface of the skull. A custom stainless steel headbar was leveled on the skull and attached using clear dental cement. After the cement had dried, targeted probe entry sites were stereotaxically marked on the cement layer with a permanent marker (Day 1: “mPFC”, 2.3AP, 0.6 ML; “somatosensory”, −0.7AP, −4.0 ML; “retrosplenial”, −2AP, −1 ML; “subiculum”, −3.5AP, 2.14 ML; Day 2: “OFC”: 2.5AP, 1.5 ML; “mPFC”: 2.3AP, −0.6 ML; “retrosplenial”, −2AP, 1 ML; “thalamus”, −1.5AP, −1.7 ML). In part, they were selected to cover areas with known connectivity to the retrosplenial cortex. A 2 mm long insulated platinum-iridium wire was implanted centrally over the cerebellum, with the last several hundred microns bared to serve as an electrical reference. Buprenorphine SR was administered to relieve pain and mice were allowed to recover for at least one week before recordings began.

Electrophysiological Recording

Mice were acclimated to head fixation and the recording rig over several days before recording. Several hours before recording, mice were anesthetized for approximately 30 minutes each while small 1 mm craniotomies were made over four pre-marked areas. Craniotomies were kept clean of bone fragments and made such that bleeding was minimized. The skull was covered with a pool of saline and with Quik-Cast to protect and keep moist craniotomies before recording. Mice recovered in their homecages before recording. Immediately prior to recording set up, mice were very briefly anesthetized with isoflurane and implanted with an IV catheter system (BD Saf-T-Intima Closed IV Catheter System: 22G ×¾″, containing an integrated Y adaptor and safety lock) to their intraperitoneal cavity and affixed with glue. The microtubing had been previously loaded with a ketamine/saline solution (0.1 ml of 100 mg/ml Ketamine solution (KetaVed, VEDCO) in 2 ml of saline) and was maintained at neutral pressure using a syringe attached at the distal (from the animal) end of the fluid line. While still anesthetized, mice were headfixed and tubing was secured stably to a custom mouse holder by tape. Quik-Cast was removed from the skull, and craniotomies were briefly cleaned before being submerged in a pool of saline. Four Neuropixels 1.0 probes^(43,44), were grounded together with the mouse reference electrode, coated with red fixable dye (CM-Dil, ThermoFisher), positioned over each craniotomy, and carefully inserted at approximately 15 degrees from the vertical axis to depths of 4-5 mm at a rate of approximately 3 um/s. Following the completion of all insertions, the probes were allowed to rest for at least 10 minutes before the recording began. 30 kHz data was acquired from the four probes simultaneously using the Neuropixels control system (as described in the User Manual) and SpikeGLX software (billkarsh.github.io/SpikeGLX/). The four acquisitions were synchronized using a common squarewave signal recorded for all probes and aligned to the nearest pulse edge. Following the beginning of recording, a 20-minute baseline period of activity was collected. A 50 mg/kg dose of ketamine was then remotely injected i.p. through the catheter system, the administration time was marked, and an additional 40 minutes of activity were recorded. Following recording, craniotomies were sealed and the skull was covered with Kwik-Cast. A custom MATLAB script wrapping Kilosort2⁴⁵ (github.com/MouseLand/Kilosort2) functions was used to spike sort data, using default Kilosort2 parameters. Following spike sorting with Kilosort2, clusters were manually curated in Phy (github.com/cortex-lab/phy) to separate “Noise” clusters from “Good” clusters. Clusters were considered “Noise” if they contained electrical artifacts, were not well isolated, or appeared to drift off of the probe (trend in decreasing amplitude correlated with decreasing spike rate). Subsequent analysis was performed using custom Python code.

Histology

Animals were perfused with phosphate buffered saline (PBS) and 4% paraformaldehyde (PFA) before overnight postfixation in 4% PFA at 4° C. Postfixed brains were maintained for <1 week at 4° C. in PBS until subsequent use. Brains were dehydrated in a methanol gradient and subsequently processed as described previously⁴⁶. Whole brains were imaged with horizontal optical sections on a LaVision light sheet microscope in dibenzyl ether for 488 nm autofluorescence and 532 nm CM-Dil electrode tracts. Image stacks were downsampled to 25 micron resolution and registered using autofluorescence signal to the Allen Brain Atlas CCFv3. Nonlinear registration and transformation was performed using Elastix. Transformed CM-Dil electrode tract data was traced in CCF coordinates using a custom variant of AllenCCF software (github.com/cortex-lab/allenCCF) and subsequently processed using custom Python code for tract reconstruction and location assignment. Well isolated unit locations were assigned to the electrode location with peak unit amplitude. This location was used to label a unit with the nearest reference atlas subdivision. Reconstructed tracts were manually assigned to corresponding Neuropixels probe recordings. As individual probe insertions were distant from each other and labeled by hemisphere on the day of insertion, 8 tracts per brain (from two sessions of recording per mouse) could be unambiguously assigned.

Data Analysis

Following recording time synchronization for all spike times (as described above), spikes for each “Good” unit were binned at 10 ms intervals and smoothed using a 50 ms width causal moving average filter, resulting in a smoothed spike rate. When shown, spiking data is not binned or smoothed. Correlations were computed using smoothed spike rate traces. For correlations with RSP, a single trace was computed with RSP as the mean firing rate across all units in RSP. This RSP trace was then correlated separately with the firing rate of each individual unit from that recording session. Firing rate was computed as the mean smoothed spike rate across the designated four-minute window before or after ketamine administration (either minutes −12 to −8 or +8 to +12). For plotting z-scored traces, for each trace, its mean was subtracted and it was divided by its standard deviation. To select units for showing in raster plot, units were sorted based on their correlation of their smoothed spike rate with the mean smoothed spike rate across all RSP units, and the top five units from each region were shown.

Fiber Photometry

A 405 nm and 470 nm LED illumination (Thorlabs) was combined (425 nm longpass, Thorlabs DMLP425) and coupled into a fiber optic patch cord (400 μm diameter, 0.48 NA, Doric Lenses) using a longpass dichroic (505 nm, Thorlabs DMLP505) and a fixed-focused coupler/collimator with a standard FC connector (F240FC-A, NA 0.51, f =7.9 mm)^(47,48). Each illumination channel was frequency modulated using the sync output from a lock-in amplifier (SR810 DSP, Stanford Research Systems). GCaMP fluorescence was collected through the same patchcord and delivered through a bandpass emission filter (Semrock, FF01-520/35) onto a NewFocus 2151 femtowatt silicon photoreceiver (Newport, DC Low mode). The signal from the photoreceiver was split into each lock-in amplifier, and the filtered outputs were digitized at 5 kHz using a NIDAQ (National Instruments PCIe-6353) and saved using a MATLAB (MathWorks) acquisition script.

Data Processing and Analysis for Recordings General Statistical Analysis

Sample sizes were chosen using standards in the field established in previous published studies and our lab's experience with the behavioral tests. Mean+/−s.e.m. was used to report statistics unless otherwise indicated. The statistical test used and the definition n for each analysis is listed in the Results text or figure legends. Multiple comparisons were false discovery rate corrected for by Benjamini-Hochberg correction (alpha=0.05) and is indicated in the text as “corrected p=”. No statistical methods were used to pre-determine sample size. Criteria for animal exclusion was pre-established: animals were excluded if injected virus did not express or optical fibers were mistargeted. Variances were similar between groups that were statistically compared except in a few cases where the mean and variance near zero. Effect size was calculated using either Hedge's g, or Glass's delta was used if group variances were very different.

Widefield Imaging and Fiber Photometry

Analysis was performed using custom MATLAB (MathWorks) scripts. Widefield imaging videos were registered to a 2D top-projection atlas generated from the annotated Allen Brain Atlas volume, version CCFv2, in MATLAB (MathWorks), using an affine transformation computed from four manually selected control points. Each video was DF/F normalized, using the median for each pixel over the entire time series as F. The 410 nm channel was then temporally smoothed using a moving average (width=400 ms). The smoothed 410 nm DF/F signal for each pixel was regressed onto the 488 nm DF/F signal for the corresponding pixel, and the regression coefficients were used to scale the 410 nm channel to the 488 nm channel. This scaled 410 nm DF/F signal was then subtracted from the 488 nm DF/F signal to produce a normalized signal for each pixel. For quantification, activity traces were extracted from points centered in five cortical regions (visual, somatosensory, parietal, motor, retrosplenial) on the left hemisphere of the brain. The video data was Gaussian smoothed spatially (radius=2 pixels). Similarly, for fiber photometry recordings, the 410 nm signal was regressed onto the 488 nm signal, the 410 nm signal was scaled by the regression coefficients, and then subtracted from the 488 nm signal.

Two Photon Imaging

Two-photon tiff stack videos were registered to the average image using the TurboReg ImageJ plug-in using rigid body transformations. Cellular region of interests (ROIs) were manually selected, since automated algorithms did not perform well in detecting cells with oscillatory activity. Contaminating neuropil signal was estimated from an annulus surrounding each soma mask and removed using the function F(t)=Fsoma(t)−0.3*Fneuropil(t), where Fsoma(t) is the somatic DF/F and Fneuropil(t) is the DF/F from an annulus 4 pixels wide surrounding each soma mask. The resulting traces were detrended and z-scored. The full frame fluorescence (used for quantifying layer 1 neuropil oscillations) was computed by summing all of the pixels in each frame on each time point, and then computing DF/F the same way, with no neuropil subtraction step.

Frequency Analysis

Each trace (corresponding to either a fiber photometry signal, a cellular two-photon signal, a widefield regional centroid, a widefield pixel, an sEEG trace, or a smoothed spike rate Neuropixels trace) was z-scored, and the power spectral density (PSD) was then estimated using Welch's method (pwelch( ) in Matlab), with a window size of 10*fs (15*fs for human sEEG) where fs is the sampling rate of the signal. The average band power within a specified band (i.e. 1-3 Hz) was then computed by integrating the PSD estimate (bandpower in Matlab). For displaying the traces of individual cells, traces were each zscored by subtracting the mean value of the trace (across the whole recorded time window) and dividing by the standard deviation of the trace. Unless otherwise indicated, the PSD was computed across the time window from minutes 10 to 11 post-injection.

Electrophysiology 32-Channel Acute Silicon Probe Recordings

Extracellular electrophysiological data were recorded using 32 channel acute silicon probes (ASSY-37 P-1, Cambridge Neurotech) acquired at 30 kHz. Following common average referencing, well-isolated single units were identified using KiloSort and Phy. Data were analyzed using custom Matlab scripts. Instantaneous firing rate was computed using 1 ms bins. We used a Hidden Markov Model with two states to label ON and OFF states. ON states were considered to represent bursts, and OFF states were inter-burst intervals. We fit the emission and transition parameters of the model using the Baum-Welch algorithm (Matlab hmmtrain with a convergence threshold of 1 e-6 and initial guesses of transition matrix: [0.95, 0.05; 0.05, 0.95] and emission: [0.5, 0.5; 0.1, 0.99]), and then estimated the state assignment at each time point using the Viterbi algorithm (Matlab hmmviterbi). Various initial guesses were tested, and they yielded the same or similar model fit. For population analyses, a bin size of 2 was used. In FIG. 10 , we are looking at the number of spikes in each burst, which can vary depending on the neuron being recorded, so we included every burst for every neuron. To quantify the consistency of the sequential onset of activation across bursts, we ranked each unit by its onset time for each burst. We then ordered the units according to their median rank across bursts. For each burst, we fit a linear model between the ranking in that burst and the median rank. The distribution of correlation coefficients was then plotted across bursts for each mouse and treatment condition.

Rodent behavior

General Behavioral Notes: Mice were handled and acclimated to patch cord coupling before testing. Opsin and control mice were randomly distributed across group-housed experiment cages. Experimenters were blinded to animal identity during performance and scoring of each behavioral assay, as well as when assessing viral expression and fiber placement. For all statistical tests between groups and involving multiple comparisons, a Benjamini-Hochberg false discovery rate correction was used.

Hot Plate Test: Mice were placed on the hot plate (Bioseb), which was set to 55° C. and surrounded by a clear circular perimeter. Mouse activity was recorded using two cameras from different angles. The experiment ended when the mouse jumped to escape or 90 seconds had elapsed. Care was taken to clean and dry the hot plate apparatus between animals. For optogenetic experiments, mice were connected to patch cords and then returned to a holding cage. Illumination began 30 seconds before mice were placed on the hot plate.

Tail Suspension Test: Mouse activity was recorded using a 60 Hz web camera. Struggling activity was quantified for minutes 2-10. For ketamine experiments, tail suspension began 5 minutes after injection, so that post injection minutes 7-15 were used for scoring. For optogenetic experiments, mice were connected to patch cords and then returned to a holding cage. Illumination began 30 seconds before tails were secured with tape and suspended from the beam. Mice were excluded if they climbed on their own tails.

Social Interaction Assay: Resident-intruder social interaction was recorded for two minutes. Interactions were defined as close physical proximity behaviors, including sniffing, touching, and following. For optogenetic experiments, mice were connected to patch cords and then returned to their home cage. Illumination began 30 seconds before the same-sex intruder mouse was introduced.

Righting Reflex Assay: Mice were held at the neck and tail, rotated to supine position on a flat surface, and quickly released. Time to right was quantified as moment of release to moment when all 4 legs touched the ground. A five second threshold was used to classify righting success (less than 5 seconds) from failure (more than 5 seconds). For optogenetic experiments, mice were connected to patch cords and then returned to a holding cage. Illumination began for 30 seconds, and then the mouse was swiftly inverted onto the flat surface.

Open Field Test: Mice were placed in an evenly illuminated 50 cm×50 cm open field test box. Mouse position was captured using a webcam and Viewer software (Biobserve).

Retrosplenial optogenetics

Stereotaxic Surgery: Adult male and female Rbp4-Cre transgenic animals were bilaterally injected with either 1 μl of AAV1-nEF-DIO-eNpHR3.0-p2a-ChR2(H134R-YFP (titer 5E12, known as eNPAC 2.0, and referred to in the text as eNPAC) or AAV5-Ef1a-D10-eYFP (titer 5E12) in retrosplenial cortex (AP −3.3, ML+/−0.5, DV 0.5). A fiber was implanted above each injected area. For S1 opsin-expressing animals, the injection was made at (AP+0, ML+/−3.0, DV 0.5). Viruses were obtained from the Stanford Neuroscience Gene Vector and Virus Core. Animals were given four weeks for viral expression and recovery before experimentation.

Illumination Parameters: For blue light: 473 nm laser, 10 ms pulses, 20 Hz, 20 mW at fiber tip. For yellow light: 594 nm, continuous light, 15 mW at fiber tip. Alternating 250 ms bouts of pulsed blue light and continuous yellow light were used for oscillatory stimulation experiments. For non-rhythmic stimulation, alternating 20 Hz blue light and continuous yellow light were also used but with length of each illumination randomly selected from a uniform distribution with a minimum of 50 milliseconds and a maximum of 2 seconds. Each 30 seconds, blue light and yellow light pulse lengths were selected such that the total time allotted to blue or yellow light across 30 seconds matched that of the rhythmic group.

Subcortical Projection Optogenetics with Fiber Photometry

Stereotaxic Surgery: Adult male and female wildtype 057/B16 wild-type animals were bilaterally injected into dorsal anterior thalamus (AP −1.2, ML+/−1, DV 3.25) or subiculum (AP −3.8, ML+/−2.5, DV −2.2) with 500 nL of either AAVdj-CaMKIIa-NpHR3.0 (titer 5.15E12) or AAV8-Ef1a-eYFP (titer 5E12). Then, 500 or 600 nL of AAVdj-CaMKIIa-GCaMP6m was injected in right retrosplenial cortex (AP −3.3, ML 0.5, DV 0.5). A fiber was implanted above each injected area (with one of the subcortical fibers angled at 30 degrees). Four weeks were allowed for viral expression and recovery before experimentation.

Illumination Parameters: 594 nm, continuous light, 15 mW at each fiber tip. Eight minutes after ketamine (50 mg/kg) administration, six minutes of photometry data were recorded: two minutes of pre-illumination photometry were recorded, followed by two minutes with continuous illumination, and then two minutes of post-stimulation.

Histology: For monosynaptic input tracing, adult male Rpb4-cre animals were injected with AAV8-EF1a-DIO-TVA-p2A-oG (9E12) into retrosplenial cortex. Two weeks later, ENVA-Rabies-GFP (3E8) was injected in the same location. After five days, animals were perfused, brains fixed overnight, and sectioned into 50 μm sections; full coronal sections were imaged on a confocal microscope for cell counting.

Retrosplenial Gene Disruption and Fiber Photometry

Stereotaxic Surgery: Adult male and female HCN1^(f/f), NR1^(f/f), or wildtype C57/B16 were injected bilaterally in two retrosplenial locations (AP −3.1 and −3.4, ML+/−0.5, DV −0.5) with 1 uL of AAV8-Ef1a-mCherry-IRES-Cre (titer 5E11) and AAVdj-Ef1a-DIO-GCaMP6m (titer 1E13). A 400-micron optical fiber was implanted over one injection location. At least four weeks were allowed for viral expression and gene disruption before photometry recording. For gene knockout behavior experiments, injections of AAVdj-Ef1a-Cre were made at (AP −2.7, −3.1 and −3.5, ML+/−0.5, DV −1.35 and −0.70). For gene knockout control behavioral experiments, two retrosplenial injections of AAVdj-Efla-Cre were made in experimental animals.

Rodent Histology: After PFA-perfusion and overnight fixation in 4% PFA at 4 C., 50 μm sections were taken on a vibratome. For immunohistochemistry, sections were washed in PBST (0.3% Triton-X) for 1 h, blocked with 1% BSA for 30 minutes, and then incubated with primary antibody in 1% PBST-BSA overnight. Primary antibodies used: anti-HCN1: 1:500 of abnova MAB6651 lot MH387188, anti-NMDAR: 1:500 of Invitrogen RA5-85751, lot UF2785857C. Next, sections were washed for 30 min in 1% PBST and then incubated with secondary antibody (1:500 in PBST-BSA) for 90 min at room temperature, and then washed for 1 hour in PBST. Sections were mounted on slides and imaged using a confocal microscope.

Human Intracranial Electroencephalography (sEEG) Recordings

All clinical research was reviewed and approved by the Stanford Institutional Review Board. Informed consent was obtained from the subject prior to participation in the study protocol. The patient (participant number S19-137/SD056) was implanted with Ad-Tech (Oak Creek, WI) SEEG Depth Electrodes as part of routine Phase II monitoring for refractory epilepsy. A 10 kHz research copy of the patient's electrophysiological data was acquired over Ethernet, simultaneous with inpatient clinical recording, via a Nihon Khoden (Tokyo, Japan) JE-120A junction box as part of an EEG-1200 clinical acquisition system. For ease of processing, the 10 kHz data was decimated to 20 Hz using a 20th-order FIR filter to evaluate the 3 Hz features. Key times surrounding seizures were reviewed at 10 kHz. Video of the patient aligned to the electrophysiology with ˜100 ms precision was part of the clinical record and was reviewed to acquire patient quotes during stimulation mapping. For quantification, 10 seconds of pre-seizure data were used, z-scored, and the power spectral density (PSD) was then estimated using Welch's method (pwelch in Matlab). The average band power within a specified band (3-4 Hz) was then computed by integrating the PSD estimate (bandpower( ) in Matlab). There was still an increase in band power in PMC during pre-seizure auras relative to non-seizure periods and to other regions when using the 1-4 Hz band, but there was baseline low frequency power in all regions that made the effect of the ˜3 Hz oscillation smaller. The clinical stimulus pulse pattern used was 50 Hz biphasic stimulation for 1-2 s.

Supplementary Note 1: Clinical Interview with Epileptologist.

Seizure Description.

PATIENT: “It would probably be better to just let you know right now that there is also a beta component to my spatial disorientation where like if you were to spin me around I would lose what is up, what is down . . . just throwing that out there.”

EPILEPTOLOGIST: “If I spin you around . . . ”

PATIENT: “If I was sitting in a chair that could spin and you were to spin that chair in any direction, I would lose my understanding of . . . ”

EPILEPTOLOGIST: “Were you born like this?”

PATIENT: “No, I had a very slow seizure a few days ago and it was one the most interesting experiences of my life. It was like-kinda like this, imagine if you were just like stretching it, it was not more intense, but because I can think through my seizures, I was able to experience the whole seizure slowly . . . it was really cool”

EPILEPTOLOGIST: “Because you are also on medications as well, that could probably . . . so what happened?”

PATIENT: “So I, the first thing I noticed was that it felt like the whole world (again, I am going to use those 6 degrees of freedom I was talking to you about . . . so the three spatial dimensions and then beta x, beta y and beta z), I could feel them move and then the next thing I knew, I was feeling the emotion or feeling of where in this 3D space am I? And I went through the feeling of the process very slowly of refiguring that out, but it was like listening to another conversation in my brain.”

EPILEPTOLOGIST: “Tell me more”

PATIENT: Laughs, “I really enjoyed it . . . um, it was kind of like, um, the experiencing, like you know, ok, there are . . . I am just going to describe it like a computer because that is the best way that I know how to describe it.”

EPILEPTOLOGIST: “Yes, sure, sure”

PATIENT: “Imagine there are these two ICs speaking with each other, two components, right, but there is one data buff. Are you familiar with a data buff?”

EPILEPTOLOGIST: “I wish I were familiar, but no, consider me a complete idiot”

PATIENT: “Alright, what it does is that there is this one information highway (and what it does . . . ) and I want to speak with this component x, y and z. A and B are speaking, but they can only speak amongst this information highway that the CPU is always able to listen to. So I am listening to A and B speak with each other to try to figure out what the hell is going on and as a result, I am listening to this conversation, the CPU is trying to figure out . . . maybe I don't know what the “me” is, I don't know what that means necessarily, I personally don't believe in the idea of a soul, personally, but you know I was aware that I was listening to two parts of my brain speak to each other in a way that a third part of my brain, which I considered me, was able to listen. And what that felt like was this, it felt like a depersonalization, if you were to say . . . my friend asked me this recently, what would it feel like if someone else were to come into your head? That is exactly what it felt like . . . what I considered me shrank to this other part of me where the other parts of my brain that were talking, I stopped considering them me”

EPILEPTOLOGIST: “Interesting.”

PATIENT: “So, that's why, for example, I took a blanket because I was interested in what was going on-I threw it over my body, just to see, because I knew that when I don't feel it, I don't consider it me and immediately my legs were no longer a part of me, in the way that that part of me was thinking, that part of my brain that I considering me was thinking so it was kind of like I was closed off, but I could hear the conversation-just like if you two were talking and you were saying “No we're upside down” and she was saying “No we're left side up”, you know, I was listening to that and getting information that you two could not get so that's why I would do things like grab or throw something so that these two could say “oh look that just fell” and to try to convince them that what I am getting from here . . . it was really weird, but I thought you would find it interesting.”

Supplementary Note 2

One characteristic of seizures is increased synchrony. While the ketamine-induced rhythm involved increased synchrony in neuronal firing, the initiation, spread, maintenance/termination, and behavior were distinguishable from typical seizures. Seizures display abrupt transitions in activity at onset and exhibit uncontrolled, variable duration; in contrast, the ketamine-induced oscillation emerged gradually over 2 minutes and decayed reliably and predictably over ˜¹⁵ minutes. Moreover, while seizures spread from the focus to other areas and across layers 49, the ketamine-rhythm did not spread beyond layer 5 neurons in retrosplenial cortex—neither to other layers nor to nearby cortical regions; cells at the periphery of the retrosplenial cortex were not recruited later, and ketamine-induced activity did not spread laterally to visual cortex, and no rigidity, convulsive movements, absence-like states, or salivation/rearing/falling were observed in response to dissociative doses of ketamine.

REFERENCES

-   1. Ferezou, I. et al. Spatiotemporal dynamics of cortical     sensorimotor integration in behaving mice. Neuron 56, 907-23 (2007). -   2. Mohajerani, M. H. et al. Spontaneous cortical activity alternates     between motifs defined by regional axonal projections. Nat.     Neurosci. 16, 1426 (2013). -   3. Musall, S., Kaufman, M. T., Juavinett, A. L., Gluf, S. &     Churchland, A. K. Single-trial neural dynamics are dominated by     richly varied movements. Nat. Neurosci. 22, 1677-1686 (2019). -   4. Kauvar, I. V. et al. Cortical Observation by Synchronous     Multifocal Optical Sampling Reveals Widespread Population Encoding     of Actions. Neuron 107, 351-367.e19 (2020). -   5. Guo, Z. V et al. Flow of cortical activity underlying a tactile     decision in mice. Neuron 81, 179-194 (2014). -   6. Wekselblatt, J. B., Flister, E. D., Piscopo, D. M. & Niell, C. M.     Large-scale imaging of cortical dynamics during sensory perception     and behavior. J. Neurophysiol. 115, 2852-2866 (2016). -   7. Ma, Y. et al. Resting-state hemodynamics are spatiotemporally     coupled to synchronized and symmetric neural activity in excitatory     neurons. Proc. Natl. Acad. Sci. U.S.A 113, E8463—E8471 (2016). -   8. Allen, W. E. et al. Global Representations of Goal-Directed     Behavior in Distinct Cell Types of Mouse Neocortex. Neuron 94,     891-907.e6 (2017). -   9. Makino, H. et al. Transformation of Cortex-wide Emergent     Properties during Motor Learning. Neuron 94, 880-890.e8 (2017). -   10. Chen, T.-W., Li, N., Daie, K. & Svoboda, K. A Map of     Anticipatory Activity in Mouse Motor Cortex. Neuron 94, 866-879.e4     (2017). -   11. Xiao, D. et al. Mapping cortical mesoscopic networks of single     spiking cortical or sub-cortical neurons. Elife 6, (2017). -   12. Gilad, A., Gallero-Salas, Y., Groos, D. & Helmchen, F.     Behavioral Strategy Determines Frontal or Posterior Location of     Short-Term Memory in Neocortex. Neuron 99, 814-828.e7 (2018). -   13. American Psychiatric Association. Diagnostic and statistical     manual of mental disorders (5th ed.). (2013). -   14. Krystal, J. H. et al. Subanesthetic Effects of the     Noncompetitive NMDA Antagonist, Ketamine, in Humans:     Psychotomimetic, Perceptual, Cognitive, and Neuroendocrine     Responses. Arch. Gen. Psychiatry 51, 199-214 (1994). -   15. Guo, Z. V et al. Procedures for behavioral experiments in     head-fixed mice. PLoS One 9, (2014). -   16. Gil-Sanz, C. et al. Lineage Tracing Using Cux2-Cre and     Cux2-CreERT2 Mice. Neuron 86, 1091-1099 (2015). -   17. Gerfen, C. R., Paletzki, R. & Heintz, N. GENSAT BAC     cre-recombinase driver lines to study the functional organization of     cerebral cortical and basal ganglia circuits. Neuron 80, 1368-83     (2013). -   18. Allen, W. E. et al. Thirst-associated preoptic neurons encode an     aversive motivational drive. Science 357, 1149-1155 (2017). -   19. DeNardo, L. A. et al. Temporal evolution of cortical ensembles     promoting remote memory retrieval. Nat. Neurosci. 22, 460-469     (2019). -   20. Oh, S. W. et al. A mesoscale connectome of the mouse brain.     Nature 508, 207-214 (2014). -   21. Hunnicutt, B. J. et al. A comprehensive thalamocortical     projection map at the mesoscopic level. Nat. Neurosci. 17, 1276-1285     (2014). -   22. Phillips, J. W. et al. A repeated molecular architecture across     thalamic pathways. Nat. Neurosci. 22, 1925-1935 (2019). -   23. McCormick, D. A. & Pape, H. C. Properties of a     hyperpolarization-activated cation current and its role in rhythmic     oscillation in thalamic relay neurones. J. Physiol. 431, 291-318     (1990). -   24. Leresche, N., Lightowler, S., Soltesz, I.,     Jassik-Gerschenfeld, D. & Crunelli, V. Low-frequency oscillatory     activities intrinsic to rat and cat thalamocortical cells. J.     Physiol. (1991). doi:10.1113/jphysiol.1991.sp018744 -   25. Poulet, J. F. A., Fernandez, L. M. J., Crochet, S. &     Petersen, C. C. H. Thalamic control of cortical states. Nat.     Neurosci. 15, 370-372 (2012). -   26. Fogerson, P. M. & Huguenard, J. R. Tapping the Brakes: Cellular     and Synaptic Mechanisms that Regulate Thalamic Oscillations. Neuron     (2016). doi:10.1016/j.neuron.2016.10.024 -   27. MacDonald, J. F., Miljkovic, Z. & Pennefather, P. Use-dependent     block of excitatory amino acid currents in cultured neurons by     ketamine. J. Neurophysiol. 58, 251-66 (1987). -   28. Anis, N. A., Berry, S. C., Burton, N. R. & Lodge, D. The     dissociative anaesthetics, ketamine and phencyclidine, selectively     reduce excitation of central mammalian neurones by     N-methyl-aspartate. Br. J. Pharmacol. 79, 565-75 (1983). -   29. Ludwig, A., Zong, X., Jeglitsch, M., Hofmann, F. & Biel, M. A     family of hyperpolarization-activated mammalian cation channels.     Nature (1998). doi:10.1038/31255 -   30. Santoro, B. et al. Identification of a gene encoding a     hyperpolarization-activated pacemaker channel of brain. Cell (1998).     doi:10.1016/S0092-8674(00)81434-8 -   31. Vogt, B. A. & Paxinos, G. Cytoarchitecture of mouse and rat     cingulate cortex with human homologies. Brain Struct. Fund. 219,     185-192 (2014). -   32. Foster, B. L. & Parvizi, J. Direct cortical stimulation of human     posteromedial cortex. Neurology 88, 685-691 (2017). -   33. Moda-Sava, R. N. et al. Sustained rescue of prefrontal circuit     dysfunction by antidepressant-induced spine formation. Science 364,     (2019). -   34. Hua, T. et al. General anesthetics activate a potent central     pain-suppression circuit in the amygdala. Nat. Neurosci. (2020).     doi:10.1038/s41593-020-0632-8 -   35. Yang, Y. et al. Ketamine blocks bursting in the lateral habenula     to rapidly relieve depression. Nature 554, 317-322 (2018). -   36. Tomitaka, M., Tomitaka, S., Rajdev, S. & Sharp, F. R. Fluoxetine     prevents PCP- and MK801-induced HSP70 expression in injured limbic     cortical neurons of rats. Biol. Psychiatry 47, 836-841 (2000). -   37. Olney, J. W., Labruyere, J. & Price, M. T. Pathological changes     induced in cerebrocortical neurons by phencyclidine and related     drugs. Science (80.). 244, 1360-1362 (1989). -   38. Mason, M. F. et al. Wandering minds: the default network and     stimulus-independent thought. Science (80.). 315, 393-395 (2007). -   39. Raichle, M. E. The Brain's Default Mode Network. Annu. Rev.     Neurosci. 38, 433-447 (2015). -   40. Kohrs, R. & Durieux, M. E. Ketamine: teaching an old drug new     tricks. Anesth. Analg. 87, 1186-1193 (1998). -   41. Green, S. M., Roback, M. G., Kennedy, R. M. & Krauss, B.     Clinical Practice Guideline for Emergency Department Ketamine     Dissociative Sedation: 2011 Update. Ann. Emerg. Med. 57, 449-461     (2011). -   42. Schwenk, E. S. et al. Consensus Guidelines on the Use of     Intravenous Ketamine Infusions for Acute Pain Management From the     American Society of Regional Anesthesia and Pain Medicine, the     American Academy of Pain Medicine, and the American Society of     Anesthesiologists. Reg. Anesth. Pain Med. 43, 456 (2018). -   43. Jun, J. J. et al. Fully integrated silicon probes for     high-density recording of neural activity. Nature 551, 232-236     (2017). -   44. Putzeys, J. et al. Neuropixels Data-Acquisition System: A     Scalable Platform for Parallel Recording of 10     000+Electrophysiological Signals. IEEE Trans. Biomed. Circuits Syst.     13, 1635-1644 (2019). -   45. Stringer, C. et al. Spontaneous behaviors drive     multidimensional, brainwide activity. Science 364, 255 (2019). -   46. Allen, W. E. et al. Thirst regulates motivated behavior through     modulation of brainwide neural population dynamics. Science 364, 253     (2019). -   47. Zalocusky, K. A. et al. Nucleus accumbens D2R cells signal prior     outcomes and control risky decision-making. Nature 531, 642-646     (2016). -   48. Gunaydin, L. a. et al. Natural neural projection dynamics     underlying social behavior. Cell 157, 1535-1551 (2014). -   49. Wenzel, M., Hamm, J. P., Peterka, D. S. & Yuste, R. Acute focal     seizures start as local synchronizations of neuronal ensembles. J.     Neurosci. 3176-18 (2019). doi:10.1523/JNEUROSCI.3176-18.2019

Example 2 Neuromodulation for Treatment of Dissociation

Acute or chronic neuromodulation of the posteromedial cortical regions (including posterior cingulate, precuneus, and retrosplenial) or thalamic nuclei for dissociative disorders is used for treatment of dissociation. Neuromodulation can be achieved using electrical stimulation from chronically implanted, clinically approved electrodes, transcranial magnetic stimulation, or focused ultrasound, among other techniques. For electrical stimulation, FDA approved deep brain stimulators are used. Cortical layers or cell-types can be targeted specifically using genetic methods, and then combined with genetically encodable modulation techniques, such as optogenetics. Open loop modulation is used to prevent future dissociative episodes by constraining neural activity patterns. Closed loop modulation is used to alleviate acute symptoms of dissociation, triggered by neural activity (e.g. oscillations) or experienced symptoms by the patient.

Example 3 Neuromodulation for Induction of Dissociation

Dissociation may be an adaptive mechanism to manage traumatic situations, anxiety, panic, depression, or chronic pain. In certain scenarios, it is beneficial to induce dissociation, and to do so without use of drugs such as ketamine. Dissociation may play a role in ketamine's efficacy as a fast-acting antidepressant. Targeted rhythmic circuit activity is induced using neuromodulation to elicit dissociation or reduce association.

Example 4 Management of Dissociation or Disorders of Association

Expression levels of rhythm-generating channels (e.g. HCN1) in the posteromedial cortex can be evaluated by immunohistochemical or other pathology assessment such as by biopsy or other labeling method, invasive or non-invasive, for clinical use in diagnosis or management of dissociation or disorders of association. 

What is claimed is:
 1. A method of inhibiting development of a dissociative state in a subject, the method comprising inhibiting a retrosplenial cortex layer-5 neuron optogenetically, wherein rhythmic neural activity is inhibited in the posteromedial cortex of the subject.
 2. The method of claim 1, wherein said inhibiting the retrosplenial cortex layer-5 neuron optogenetically comprises: introducing a recombinant polynucleotide encoding a light-responsive ion channel into the retrosplenial cortex layer-5 neuron, wherein the light-responsive ion channel is expressed in the retrosplenial cortex layer-5 neuron; and illuminating the light-responsive ion channel with light at a wavelength that activates the light-responsive ion channel, wherein conduction of ions by the light-responsive ion channel in response to absorption of light results in hyperpolarization and inhibition of the retrosplenial cortex layer-5 neuron.
 3. The method of claim 2, wherein the light-responsive ion channel is a light-responsive anion-conducting opsin or a light-responsive proton conductance regulator.
 4. The method of claim 3, wherein the light-responsive anion-conducting opsin conducts chloride ions (Cl⁻).
 5. The method of claim 3 or 4, wherein the anion-conduction opsin is an anion-conducting channelrhodopsin or halorhodopsin.
 6. The method of claim 5, wherein the halorhodopsin is a Natronomonas pharaonis halorhodopsin (NpHR), enhanced NpHR (eNpHR) 1.0, eNpHR 2.0, or eNpHR 3.0.
 7. The method of claim 5, wherein the anion-conducting channelrhodopsin is iC1C2, SwiChR, SwiChR++, or iC++.
 8. The method of claim 3, wherein the light-responsive proton conductance regulator is a bacteriorhodopsin or an archaerhodopsin.
 9. The method of claim 8, wherein the light-responsive proton conductance regulator is Arch from Halorubrum sodomense, ArchT from Halorubrum sp., TP009 from Leptosphaeria maculans, or Mac from Leptosphaeria maculans.
 10. The method of any one of claims 2 to 9, wherein the polynucleotide encoding the light-responsive ion channel is provided by a viral vector.
 11. The method of claim 10, wherein the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector.
 12. The method of claim 10 or 11, wherein the viral vector is stereotactically injected into the retrosplenial cortex.
 13. The method of any one of claims 10 to 12, wherein the vector further comprises a neuron-specific promoter operably linked to the polynucleotide encoding the light-responsive ion channel.
 14. The method of any one of claims 10 to 13, wherein expression of the light-responsive ion channel is inducible.
 15. The method of any one of claims 1 to 14, wherein said illuminating the light-responsive ion channel comprises delivering light from a light source to the light-responsive ion channel using a fiber-optic-based optical neural interface.
 16. The method of claim 15, wherein the light source is a solid-state diode laser.
 17. The method of any one of claims 1 to 16, wherein the dissociative disorder is caused by a drug, a seizure, childhood psychological or physical trauma, abuse, sexual assault, or a dissociation-linked neuropsychiatric disorder.
 18. The method of claim 17, wherein the dissociation-linked neuropsychiatric disorder is post-traumatic stress disorder (PTSD), borderline personality disorder, or schizophrenia spectrum disorder.
 19. A method of inducing dissociative symptoms in a subject, the method comprising performing rhythmic optogenetic activation of a retrosplenial cortex layer-5 neuron, wherein rhythmic neural activity is induced in the posteromedial cortex of the subject.
 20. The method of claim 19, wherein said performing rhythmic optogenetic activation of a retrosplenial cortex layer-5 neuron comprises: introducing a recombinant polynucleotide encoding a light-responsive ion channel into the retrosplenial cortex layer-5 neuron, wherein the light-responsive ion channel is expressed in the retrosplenial cortex layer-5 neuron; and rhythmically illuminating the light-responsive ion channel with light at a wavelength that activates the light-responsive ion channel, wherein conduction of ions by the light-responsive ion channel in response to absorption of light results in depolarization and activation of the retrosplenial cortex layer-5 neuron.
 21. The method of claim 20, wherein the light-responsive ion channel is a light-responsive cation-conducting opsin.
 22. The method of claim 21, wherein the light-responsive cation-conducting opsin conducts calcium cations (Ca²⁺).
 23. The method of claim 21 or 22, wherein the light-responsive cation-conducting opsin is a light-responsive cation-conducting channelrhodopsin.
 24. The method of claim 23, wherein the light-responsive cation-conducting channelrhodopsin is a Chlamydomonas reinhardtii channelrhodopsin or a Volvox carteri channelrhodopsin.
 25. The method of claim 24, wherein the light-responsive cation-conducting channelrhodopsin is a Chlamydomonas reinhardtii channelrhodopsin-1 (ChR1), a Chlamydomonas reinhardtii channelrhodopsin-2 (ChR2), a Volvox carteri channelrhodopsin-1 (VChR1), or a chimeric ChR1-VChR1 channelrhodopsin.
 26. The method of any one of claims 20 to 25, wherein the polynucleotide encoding the light-responsive ion channel is provided by a viral vector.
 27. The method of claim 26, wherein the viral vector is a lentiviral vector or an adeno-associated viral (AAV) vector.
 28. The method of claim 26 or 27, wherein the viral vector is stereotactically injected into the retrosplenial cortex.
 29. The method of any one of claims 26 to 28, wherein the vector further comprises a neuron-specific promoter operably linked to the polynucleotide encoding the light-responsive ion channel.
 30. The method of any one of claims 26 to 29, wherein expression of the light-responsive ion channel is inducible.
 31. The method of any one of claims 20 to 30, wherein said rhythmically illuminating the light-responsive ion channel comprises delivering light from a light source to the light-responsive ion channel using a fiber-optic-based optical neural interface.
 32. The method of claim 31, wherein the light source is a solid-state diode laser.
 33. The method of any one of claims 20 to 32, wherein said rhythmically illuminating the light-responsive ion channel with light comprises repeatedly delivering light at the wavelength that activates the light-responsive ion channel with a frequency of 2 Hz.
 34. The method of any one of claims 20 to 33, wherein said rhythmically illuminating the light-responsive ion channel with light comprises alternating between a stimulation interval and a non-stimulation interval, wherein the light at the wavelength that activates the light-responsive ion channel is delivered during the stimulation interval, and no light or light that is not at the wavelength that activates the light-responsive ion channel or that inhibits the light-responsive ion channel is delivered during the non-stimulation interval.
 35. The method of claim 34, wherein the stimulation interval is 100 milliseconds to 300 milliseconds.
 36. The method of claim 35, wherein the stimulation interval is 250 milliseconds.
 37. The method of any one of claims 34 to 36, wherein the non-stimulation interval is 100 milliseconds to 300 milliseconds.
 38. The method of any one of claims 19 to 37, wherein the subject has a disorder of association.
 39. The method of claim 38, wherein the disorder of association is chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction, or dependency.
 40. A method of inducing dissociative symptoms in a subject, the method comprising: positioning an electrode to deliver electrical stimulation to a posteromedial cortex region of the brain of the subject; and applying electrical stimulation to the posteromedial cortex region using the electrode in a manner effective to generate rhythmic neural activity in the posteromedial cortex of the subject.
 41. The method of claim 40, wherein the method comprises applying electrical stimulation to a right posteromedial cortex region or a left posteromedial cortex region.
 42. The method of claim 41, wherein the electrode is placed on a surface of the right posteromedial cortex region or the left posteromedial cortex region.
 43. The method of any one of claim 41, wherein the electrode is placed within the right posteromedial cortex region or the left posteromedial cortex region.
 44. The method of any one of claims 40 to 43, wherein said applying electrical stimulation comprises delivering electrical stimulation to a posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the subject.
 45. The method of any one of claims 40 to 44, wherein the electrode is a non-brain penetrating surface electrode array or a brain-penetrating electrode array.
 46. The method of any one of claims 40 to 45, wherein the subject has a disorder of association.
 47. The method of claim 46, wherein the disorder of association is chronic pain, depression, anxiety, obsessive-compulsive disorder, addiction, or dependency.
 48. The method of any one of claims 40 to 47, wherein said electrical stimulation is applied at 2 mA to 10 mA.
 49. The method of any one of claims 40 to 48, wherein said electrical stimulation is applied for up to 2 seconds.
 50. The method of claim 49, wherein said electrical stimulation is applied for 1 second to 2 seconds.
 51. The method of any one of claims 40 to 50, wherein the electrical stimulation is applied unilaterally or bilaterally.
 52. The method of any one of claims 40 to 51, wherein the electrical stimulation is applied with a frequency of 50 Hz.
 53. The method of any one of claims 40 to 52, further comprising mapping the brain of the subject to identify an optimal location in the posteromedial cortex for positioning the electrode for delivery of the electrical stimulation to induce dissociative symptoms in the subject.
 54. The method of any one of claims 52 to 55, wherein the electrical stimulation is delivered rhythmically.
 55. A method of modulating associative or dissociative symptoms in a subject, the method comprising delivering electrical stimulation to a posteromedial cortex region of the brain of the subject that modulates rhythmic neural activity in the posteromedial cortex of the subject.
 56. The method of claim 55, wherein the electrical stimulation is delivered optogenetically, with an electrode, or by transcranial magnetic stimulation.
 57. The method of any one of claim 56, wherein the electrode is a non-brain penetrating surface electrode array or a brain-penetrating electrode array.
 58. The method of claims 55 to 57, wherein the electrical stimulation is delivered to a posterior cingulate, precuneus, or retrosplenial region of the posteromedial cortex of the subject.
 59. The method of any one of claims 55 to 58, wherein the electrical stimulation is delivered rhythmically or continuously during a stimulation period.
 60. The method of any one of claims 55 to 59, further comprising mapping the brain of the subject to identify an optimal location in the posteromedial cortex for positioning the electrode for delivery of the electrical stimulation to modulate associative or dissociative symptoms in the subject.
 61. The method of any one of claims 55 to 60, wherein the electrical stimulation is applied unilaterally or bilaterally. 